Rapid-acting insulin formulation comprising a substituted anionic compound

ABSTRACT

A composition in aqueous solution includes insulin and at least one substituted anionic compound chosen from substituted anionic compounds consisting of a backbone formed from a discrete number u of between 1 and 8 (1≤u≤8) of identical or different saccharide units, linked via identical or different glycoside bonds, the saccharide units being chosen from the group consisting of hexoses, in cyclic form or in open reduced form, said compound comprising partially substituted carboxyl functional groups, the unsubstituted carboxyl functional groups being salifiable. A pharmaceutical formulation including the composition is also set forth.

The present application is a continuation of U.S. patent applicationSer. No. 16/687,339 filed on Nov. 18, 2019, which is a continuation ofapplication Ser. No. 15/609,670 filed May 31, 2017, which is acontinuation of application Ser. No. 14/079,516 filed Nov. 13, 2013,which claims the benefit of and priority to U.S. provisional ApplicationNo. 61/726,349 filed Nov. 14, 2012; U.S. provisional Application No.61/725,775 filed Nov. 13, 2012; French Patent Application No. FR12/60855 filed Nov. 14, 2012; and French Patent Application No. FR12/60808 filed Nov. 13, 2012. Each of the prior applications isincorporated herein by reference in its entirety.

The present invention relates to a rapid-acting insulin formulation.

Since the production of insulin by genetic engineering, at the start ofthe 1980s, diabetic patients have benefited from human insulin for theirtreatment. This product has greatly improved this therapy, since theimmunological risks associated with the use of non-human insulin, inparticular pig insulin, are eliminated. However, subcutaneously-injectedhuman insulin has a hypoglycemiant effect only after 60 minutes, whichmeans that diabetic patients treated with human insulin must take theinjection 30 minutes before a meal.

One of the problems to be solved for improving the health and comfort ofdiabetic patients is that of providing them with insulin formulationswhich afford a faster hypoglycemiant response than that of humaninsulin, if possible a response approaching the physiological responseof a healthy person. The secretion of endogenous insulin in a healthyindividual is immediately triggered by the increase in glycemia. Theobject is to minimize the delay between the injection of insulin and thestart of the meal.

At the present time, it is accepted that the provision of suchformulations is useful in order for the patient to receive the bestpossible health care.

Genetic engineering has made it possible to afford a response with thedevelopment of rapid insulin analogs. These insulins are modified on oneor two amino acids so as to be more rapidly absorbed into the bloodcompartment after a subcutaneous injection. These insulins lispro(Humalog®, Lilly), aspart (Novolog®, Novo) and glulisine (Apidra®,Sanofi Aventis) are stable insulin solutions with a fasterhypoglycemiant response than that of human insulin. Consequently,patients treated with these rapid insulin analogs can take an insulininjection only 15 minutes before a meal.

The principle of rapid insulin analogs is to form hexamers at aconcentration of 100 IU/mL to ensure the stability of the insulin in thecommercial product while at the same time promoting very rapiddissociation of these hexamers into monomers after subcutaneousinjection so as to obtain rapid action.

Human insulin as formulated in its commercial form does not make itpossible to obtain a hypoglycemiant response close in kinetic terms tothe physiological response generated by the start of a meal (increase inglycemia), since, at the working concentration (100 IU/mL), in thepresence of zinc and other excipients such as phenol or m-cresol, itassembles to form a hexamer, whereas it is active in monomer and dimerform. Human insulin is prepared in the form of hexamers so as to bestable for up to 2 years at 4° C., since, in the form of monomers, ithas a very high propensity to aggregate and then to fibrillate, whichmakes it lose its activity. Furthermore, in this aggregated form, itpresents an immunological risk to the patient.

The dissociation of the hexamers into dimers and of the dimers intomonomers delay its action by close to 20 minutes when compared with arapid insulin analog (Brange J., et al., Advanced Drug Delivery Review,35, 1999, 307-335).

In addition, the kinetics of passage of the insulin analogs into theblood and their glycemia reduction kinetics are not optimal, and thereis a real need for a formulation which has an even shorter action timein order to come close to the kinetics of endogenous insulin secretionin healthy individuals.

The company Biodel has proposed a solution to this problem with a humaninsulin formulation comprising EDTA and citric acid as described inpatent application US 200839365. The capacity of EDTA to complex zincatoms and the interactions of citric acid with the cationic regionspresent at the surface of insulin are described as destabilizing thehexameric form of insulin and thus as reducing its action time.

However, such a formulation especially has the drawback ofdisassociating the hexameric form of insulin, which is the only stableform capable of meeting the stability requirements of the pharmaceuticalregulations.

PCT patent application WO 2010/122385 in the name of the Applicant isalso known, describing human insulin or insulin analog formulations thatcan solve the various problems mentioned above via the addition of asubstituted polysaccharide comprising carboxyl groups.

However, the requirements entailed by the chronic and intensive use oreven the pediatric use of such formulations lead a person skilled in theart to seek to use excipients whose molar mass and size are as small aspossible in order to facilitate their elimination.

The polysaccharides described in patent applications WO 2010/122385A1and US 2012/094902A1 as excipients are compounds consisting of chainswhose lengths are statistically variable and which are very rich inpossible sites of interaction with protein active principles. Thisrichness might induce a lack of specificity in terms of interaction, anda smaller and better defined molecule might make it possible to be morespecific in this respect.

In addition, a molecule with a well-defined backbone is generally moreeasily traceable (for example MS/MS) in biological media duringpharmacokinetic or ADME (administration, distribution, metabolism,elimination) experiments when compared with a polymer which generallygives a very diffuse and noisy signal in mass spectrometry.

Conversely, it is not excluded that a well-defined and shorter moleculemight have a deficit of possible sites of interaction with proteinactive principles. Specifically, on account of their reduced size, theydo not have the same properties as polymers of polysaccharide type sincethere is loss of the polymer effect, as is demonstrated in thecomparative examples of the experimental section; see especially thetests of insulin dissolution at the isoelectric point and the tests ofinteraction with a model protein such as albumin.

Despite these discouraging results, the Applicant has succeeded indeveloping formulations that are capable of accelerating insulin byusing a substituted anionic compound in combination with a polyanioniccompound.

Furthermore, as in the case of the use of polysaccharides, the hexamericnature of insulin is not affected, and thus the stability of theformulations is not affected, as is moreover confirmed by the examplesof state of association of human insulin and insulin analog via circulardichroism in the presence of a substituted anionic compound according tothe invention.

The present invention makes it possible to solve the various problemsoutlined above since it makes it possible especially to prepare a humaninsulin or insulin analog formulation, which is capable, afteradministration, of accelerating the passage of the human insulin or ofanalogs thereof into the blood and of more quickly reducing glycemiawhen compared with the corresponding commercial insulin products.

Following is a brief description of the drawings.

FIG. 1: DGlucose (mM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B8, glucoseTmin=30±11 min, curve plotted with the triangles corresponding toexample B2, glucose Tmin=44±14 min.

FIG. 2: DInsulin (pM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B8, insulinTmax=11±6 min, curve plotted with the triangles corresponding to exampleB2, insulin Tmax=18±8 min.

FIG. 3: DGlucose (mM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B10, glucoseTmin=33±13 min, curve plotted with the triangles corresponding toexample B2, glucose Tmin=44±14 min.

FIG. 4: DInsulin (pM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B10, insulinTmax=15±9 min, curve plotted with the triangles corresponding to exampleB2, insulin Tmax=18±8 min.

FIG. 5: DGlucose (mM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B7, glucoseTmin=41±16 min, curve plotted with the triangles corresponding toexample B2, glucose Tmin=50±14 min.

FIG. 6: DInsulin (pM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B2, insulinTmax=21±10 min, curve plotted with the triangles corresponding toexample B2, insulin Tmax=20±9 min.

FIG. 7: DGlucose (mM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B3, glucoseTmin=61±31 min, curve plotted with the triangles corresponding toexample B1, glucose Tmin=44±13 min.

FIG. 8: DInsulin (pM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B3, insulinTmax=36±33 min, curve plotted with the triangles corresponding toexample B1, insulin Tmax=28±13 min.

FIG. 9: DGlucose (mM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B39, glucoseTmin=46±9 min, curve plotted with the triangles corresponding to exampleB1, glucose Tmin=53±24 min.

FIG. 10: DInsulin (pM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B39, insulinTmax=20±7 min, curve plotted with the triangles corresponding to exampleB1, insulin Tmax=22±10 min.

FIG. 11 describes on the x-axis, from left to right:

-   -   Humalog    -   Humalog+9.3 mM citrate    -   Humalog+6 mM EDTA    -   Humalog+6 mM EDTA+9.3 mM citrate    -   Humalog+7.3 mg/ml Compound 1    -   Humalog+7.3 mg/ml Compound 1+9.3 mM citrate    -   Humalog+7.3 mg/ml Compound 1+20 mg/ml Polyanionic compound 1 and        on the y-axis the CD signal at 251 nm (deg·cm²·dmol⁻¹).

FIG. 12 describes on the x-axis, from left to right:

-   -   rhINS    -   rhINS+9.3 mM citrate    -   rhINS+6 mM EDTA    -   rhINS+6 mM EDTA+9.3 mM citrate    -   rhINS+7.3 mg/ml Compound 1    -   rhINS+7.3 mg/ml Compound 1+9.3 mM citrate    -   rhINS+7.3 mg/ml Compound 1+20 mg/ml Polyanionic compound 1 and        on the y-axis the CD signal at 275 nm (deg·cm²·dmol⁻¹).

FIG. 13: DGlucose (mM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B11, glucoseTmin=32±10 min, curve plotted with the triangles corresponding toexample B2, glucose Tmin=41±21 min.

FIG. 14: DInsulin (pM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B11, insulinTmax=13±5 min, curve plotted with the triangles corresponding to exampleB2, insulin Tmax=22±13 min.

FIG. 15: DGlucose (mM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B102, glucoseTmin=47±30 min, curve plotted with the triangles corresponding toexample B1, glucose Tmin=47±15 min.

FIG. 16: DInsulin (pM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B38, insulinTmax=22±21 min, curve plotted with the triangles corresponding toexample B1, insulin Tmax=19±12 min.

FIG. 17: DGlucose (mM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B53, glucoseTmin=63±36 min, curve plotted with the triangles corresponding toexample B1, glucose Tmin=53±19 min.

FIG. 18: DInsulin (pM) as a function of time after injection (min.).Curve plotted with the squares corresponding to example B53, insulinTmax=19±12 min, curve plotted with the triangles corresponding toexample B1, insulin Tmax=19±6 min.

FIG. 19 describes on the x-axis:

-   -   A: lispro insulin (100 IU/mL)    -   B: lispro insulin+7.3 mg/mL of compound 2    -   C: lispro insulin+7.3 mg/mL of compound 2+citrate at 9.3 mM    -   D: lispro insulin+7.3 mg/mL of compound 1    -   E: lispro insulin+7.3 mg/mL of compound 1+citrate at 9.3 mM    -   F: lispro insulin+7.3 mg/mL of compound 3    -   G: lispro insulin+7.3 mg/mL of compound 3+citrate at 9.3 mM    -   H: lispro insulin+7.3 mg/mL of compound 4    -   I: lispro insulin+7.3 mg/mL of compound 4+citrate at 9.3 mM    -   J: lispro insulin+7.3 mg/mL of compound 5    -   K: lispro insulin+7.3 mg/mL of compound 5+citrate at 9.3 mM    -   L: lispro insulin+7.3 mg/mL of compound 6    -   M: lispro insulin+7.3 mg/mL of compound 6+citrate at 9.3 mM    -   N: lispro insulin+7.3 mg/mL of compound 7    -   O: lispro insulin+7.3 mg/mL of compound 7+citrate at 9.3 mM    -   P: lispro insulin+7.3 mg/mL of compound 8    -   Q: lispro insulin+7.3 mg/mL of compound 8+citrate at 9.3 mM    -   R: lispro insulin+7.3 mg/mL of compound 9    -   S: lispro insulin+7.3 mg/mL of compound 9+citrate at 9.3 mM    -   T: lispro insulin+7.3 mg/mL of compound 10    -   U: lispro insulin+7.3 mg/mL of compound 10+citrate at 9.3 mM    -   V: lispro insulin+7.3 mg/mL of compound 11    -   W: lispro insulin+7.3 mg/mL of compound 11+citrate at 9.3 mM and        on the y-axis the circular dichroism signal at 251 nm        (deg·cm²·dmol⁻¹).

FIG. 20 describes on the x-axis:

-   -   A: human insulin (100 IU/mL)    -   B: human insulin+7.3 mg/mL of compound 2    -   C: human insulin+7.3 mg/mL of compound 2+citrate at 9.3 mM    -   D: human insulin+7.3 mg/mL of compound 1    -   E: human insulin+7.3 mg/mL of compound 1+citrate at 9.3 mM    -   F: human insulin+7.3 mg/mL of compound 3    -   G: human insulin+7.3 mg/mL of compound 3+citrate at 9.3 mM    -   H: human insulin+7.3 mg/mL of compound 4    -   I: human insulin+7.3 mg/mL of compound 4+citrate at 9.3 mM    -   J: human insulin+7.3 mg/mL of compound 5    -   K: human insulin+7.3 mg/mL of compound 5+citrate at 9.3 mM    -   L: human insulin+7.3 mg/mL of compound 6    -   M: human insulin+7.3 mg/mL of compound 6+citrate at 9.3 mM    -   N: human insulin+7.3 mg/mL of compound 7    -   O: human insulin+7.3 mg/mL of compound 7+citrate at 9.3 mM    -   P: human insulin+7.3 mg/mL of compound 8    -   Q: human insulin+7.3 mg/mL of compound 8+citrate at 9.3 mM    -   R: human insulin+7.3 mg/mL of compound 9    -   S: human insulin+7.3 mg/mL of compound 9+citrate at 9.3 mM    -   T: human insulin+7.3 mg/mL of compound 10    -   U: human insulin+7.3 mg/mL of compound 10+citrate at 9.3 mM    -   V: human insulin+7.3 mg/mL of compound 11    -   W: human insulin+7.3 mg/mL of compound 11+citrate at 9.3 mM and        on the y-axis the circular dichroism signal at 275 nm        (deg·cm²·dmol⁻¹).

The invention consists of a composition, in aqueous solution, comprisinginsulin in hexameric form, at least one substituted anionic compound anda non-polymeric polyanionic compound.

The term “substituted anionic compound” means compounds consisting of asaccharide backbone formed from a discrete number u of between 1 and 8(1≤u≤8) of identical or different saccharide units, linked via identicalor different glycoside bonds, said saccharide units being chosen fromthe group consisting of hexoses, in cyclic form or in open reduced form,said compound comprising partially substituted carboxyl functionalgroups, the unsubstituted carboxyl functional groups being salifiable.

In one embodiment, the insulin is in hexameric form.

In one embodiment, the insulin is human insulin.

The term “human insulin” means an insulin obtained by synthesis orrecombination, whose peptide sequence is the sequence of human insulin,including the allelic variations and homologs.

In one embodiment, the insulin is a recombinant human insulin asdescribed in the European Pharmacopea and the American Pharmacopea.

In one embodiment, the insulin is an insulin analog.

The term “insulin analog” means a recombinant insulin whose primarysequence contains at least one modification relative to the primarysequence of human insulin.

In one embodiment, the insulin analog is chosen from the groupconsisting of the insulin lispro (Humalog®), the insulin aspart(Novolog®, Novorapid®) and the insulin glulisine (Apidra®).

In one embodiment, the insulin analog is the insulin lispro (Humalog®).

In one embodiment, the insulin analog is the insulin aspart (Novolog®,Novorapid®).

In one embodiment, the insulin analog is the insulin glulisine(Apidra®).

In one embodiment, the substituted anionic compound is chosen fromsubstituted anionic compounds, in isolated form or as a mixture,consisting of a backbone formed from a discrete number u of between 1and 8 (1≤u≤8) of identical or different saccharide units, linked viaidentical or different glycoside bonds, said saccharide units beingchosen from the group consisting of hexoses, in cyclic form or in openreduced form, characterized in that they are substituted with:

-   -   a) at least one substituent of general formula I:        —[R₁]_(a)-[AA]_(m)  Formula I        -   the substituents being identical or different when there are            at least two substituents, in which:        -   the radical -[AA] denotes an amino acid residue,        -   the radical —R₁— being:            -   either a bond and then a=0 and the amino acid residue                -[AA] is directly linked to the backbone via a function                G,            -   or a C2 to C15 carbon-based chain, and then a=1,                optionally substituted and/or comprising at least one                heteroatom chosen from O, N and S and at least one acid                function before the reaction with the amino acid, said                chain forming with the amino acid residue -[AA] an amide                function, and is attached to the backbone by means of a                function F resulting from a reaction between a hydroxyl                function borne by the backbone and a function or                substituent borne by the precursor of the radical —R₁—,        -   F is a function chosen from ether, ester and carbamate            functions,        -   G is a carbamate function,        -   m is equal to 1 or 2,        -   the degree of substitution of the saccharide units, j, in            —[R₁]_(a)-[AA]_(m) being strictly greater than 0 and less            than or equal to 6, 0<j≤6    -   b) and, optionally, one or more substituents —R′₁,        -   the substituent —R′₁ being a C2 to C15 carbon-based chain,            which is optionally substituted and/or comprising at least            one heteroatom chosen from O, N and S and at least one acid            function in the form of an alkali metal cation salt, said            chain being linked to the backbone via a function F′            resulting from a reaction between a hydroxyl function borne            by the backbone and a function or substituent borne by the            precursor of the substituent —R′₁,        -   F′ is an ether, ester or carbamate function,        -   the degree of substitution of the saccharide units, i, in            —R′₁, being between 0 and 6−j, 0≤i≤6−j, and        -   F and F′ are identical or different,        -   F and G are identical or different,            -   i+j≤6,        -   —R′₁ is identical to or different from —R₁—,        -   the free salifiable acid functions borne by the substituent            —R′₁ are in the form of alkali metal cation salts,        -   said glycoside bonds, which may be identical or different,            being chosen from the group consisting of glycoside bonds of            (1,1), (1,2), (1,3), (1,4) or (1,6) type, in an alpha or            beta geometry.

In one embodiment, the substituted anionic compound, in isolated form oras a mixture, is chosen from substituted anionic compounds consisting ofa saccharide backbone formed from a discrete number u of between 1 and 8(1≤u≤8) of identical or different saccharide units, linked via identicalor different glycoside bonds, said saccharide units being chosen fromhexoses, in cyclic form or in open reduced form, characterized

-   -   a) in that they are randomly substituted with:        -   at least one substituent of general formula I:            —[R₁]_(a)-[AA]_(m)  Formula I    -   the substituents being identical or different when there are at        least two substituents, in which:    -   the radical -[AA]- denotes an amino acid residue, said amino        acid being chosen from the group consisting of phenylalanine,        alpha-methylphenylalanine, 3,4-dihydroxyphenylalanine, tyrosine,        alpha-methyltyrosine, O-methyltyrosine, alpha-phenylglycine,        4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine, and the        alkali metal cation salts thereof, said derivatives being of L        or D absolute configuration, -[AA] is attached to the backbone        of the molecule via a linker arm —R₁— or directly linked to the        backbone via a function G,    -   —R₁— being:        -   either a bond G, and then a=0,        -   or a C2 to C15 carbon-based chain, and then a=1, which is            optionally substituted and/or comprising at least one            heteroatom chosen from O, N and S and bearing at least one            acid function before the reaction with the amino acid, said            chain forming with the amino acid residue -[AA] an amide            bond, and is attached to the saccharide backbone via a            function F resulting from a reaction between a hydroxyl            function borne by the backbone and a function borne by the            precursor of R₁,    -   F is an ether, ester or carbamate function,    -   G is a carbamate function,    -   m is equal to 1 or 2,    -   the degree of substitution, j, in —[R₁]_(a)-[AA]_(m) being        strictly greater than 0 and less than or equal to 6, 0<j≤6,    -   and, optionally,        -   one or more substituents —R′₁    -   —R′₁ being a C2 to C15 carbon-based chain, which is optionally        substituted and/or comprising at least one heteroatom (such as        O, N and S) and bearing at least one acid function in the form        of an alkali metal cation salt, said chain being attached to the        saccharide backbone via a function F′ resulting from a reaction        between a hydroxyl function borne by the backbone and a function        borne by the precursor of —R′₁,    -   F′ is an ether, ester or carbamate function,    -   the degree of substitution, i, in —R′₁, being between 0 and 6−j,        0≤i≤6−j, and    -   —R′₁— is identical to or different from —R₁,    -   F and F′ are identical or different,    -   F′ and G are identical or different,    -   the free salifiable acid functions are in the form of alkali        metal cation salts,    -   b) said glycoside bonds, which may be identical or different,        being chosen from the group consisting of glycoside bonds of        (1,1), (1,2), (1,3), (1,4) or (1,6) type, in an alpha or beta        geometry,    -   c) i+j≤6.

In one embodiment, m is equal to 1.

In one embodiment, —R₁ and —R′₁, which may be identical or different,are a C2 to C8 carbon-based chain.

In one embodiment, —R₁ and —R′₁, which may be identical or different,are a C2 to C4 carbon-based chain.

i and j are statistical degrees of substitution and represent the meannumber of substituents per saccharide unit. Since each saccharide unitbears several hydroxyl functions of different reactivity, thedistribution of the substituents on the substituted anionic compoundsmay be different from one saccharide unit to another within the samepolyanionic compound.

In one embodiment, 0.3≤i.

In one embodiment, 0.4≤i.

In one embodiment, i≤3.

In one embodiment, i≤2.5.

In one embodiment, 0.3≤j.

In one embodiment, 0.4≤j.

In one embodiment, j≤2.

In one embodiment, j≤1.8.

In one embodiment, i and j are such that 0<i+j≤6.

In one embodiment, 0<i+j≤5.

In one embodiment, 0<i+j≤4.

In one embodiment, 0<i+j≤3.

In one embodiment, 0<i+j≤2.5.

In one embodiment, 0<i+j≤2.

In one embodiment, 0.5≤i+j≤3.

In one embodiment, 0.5≤i+j≤2.5.

In one embodiment, 0.5≤i+j≤2.

In one embodiment, 0.6≤i+j≤3.5.

In one embodiment, 0.8≤i+j≤2.5.

In one embodiment, 0.7≤i+j≤2.5.

In one embodiment, 0.7≤i+j≤2.

In one embodiment, 1<i+j≤2.5.

In one embodiment, 1<i+j≤2.

In one embodiment, —R1 and —R′1 are attached to the backbone via anether bond.

In one embodiment, when —R1- is a carbon-based chain, it is directlyattached to the backbone via an ether bond.

In one embodiment, when —R1- is a carbon-based chain, it optionallycomprises a heteroatom chosen from the group consisting of O, N and S.

In one embodiment, —R1- forms with the amino acid residue AA an amidebond, and is directly attached to the backbone via an ether function F.

In one embodiment, —R1- forms with the amino acid residue AA an amidebond, and is directly attached to the backbone via a carbamate functionF.

In one embodiment, —R1- forms with the amino acid residue AA an amidebond, and is directly attached to the backbone via an ester function F.

In one embodiment, —R1- and —R′1 are chosen from radicals of formulae IIand III

in which:

-   -   o and p, which may be identical or different, are greater than        or equal to 1 and less than or equal to 12, and    -   —R₃, —R′₃, —R₄ and —R′₄, which may be identical or different,        are chosen from the group consisting of a hydrogen atom, a        saturated or unsaturated, linear, branched or cyclic C1 to C6        alkyl, a benzyl, a C7 to C10 alkylaryl and optionally comprising        heteroatoms chosen from the group consisting of O, N and/or S,        or functions chosen from the group consisting of carboxylic        acid, amine, alcohol and thiol functions.

In one embodiment, —R1- before attachment to -AA-, is —CH₂—COOH.

In one embodiment, the substituted anionic compounds according to theinvention are characterized in that the radical —R′1 is —CH₂—COOH.

In one embodiment, —R1- before optional attachment to -AA-, is derivedfrom citric acid.

In one embodiment, —R1- before optional attachment to -AA-, is derivedfrom malic acid.

In one embodiment, —R′1 is derived from citric acid.

In one embodiment, —R′1 is derived from malic acid.

In one embodiment, —R1-, before attachment to -AA-, is chosen from thefollowing groups, in which * represents the site of attachment to F:

or the salts thereof with alkali metal cations chosen from the groupconsisting of Na⁺ and K⁺.

In one embodiment, —R′₁ is chosen from the following groups, in which *represents the site of attachment to F:

or the salts thereof with alkali metal cations chosen from the groupconsisting of Na⁺ and K⁺.

In one embodiment, the radical -[AA] is a residue of phenylalanine andof alkali metal cation salts thereof of L, D or racemic absoluteconfiguration.

In one embodiment, the radical -[AA] is a residue ofalpha-methylphenylalanine and of alkali metal cation salts thereof of L,D or racemic absolute configuration.

In one embodiment, the radical -[AA] is a residue of3,4-dihydroxyphenylalanine and of alkali metal cation salts thereof ofL, D or racemic absolute configuration.

In one embodiment, the radical -[AA] is a residue of tyrosine and ofalkali metal cation salts thereof of L, D or racemic absoluteconfiguration.

In one embodiment, the radical -[AA] is a residue ofalpha-methyltyrosine and of alkali metal cation salts thereof of L, D orracemic absolute configuration.

In one embodiment, the radical -[AA] is a residue of O-methyltyrosineand of alkali metal cation salts thereof of L, D or racemic absoluteconfiguration.

In one embodiment, the radical -[AA] is a residue of alpha-phenylglycineand of alkali metal cation salts thereof of L, D or racemic absoluteconfiguration.

In one embodiment, the radical -[AA] is a residue of4-hydroxyphenylglycine and of alkali metal cation salts thereof of L, Dor racemic absolute configuration.

In one embodiment, the radical -[AA] is a residue of3,5-dihydroxyphenylglycine and of alkali metal cation salts thereof ofL, D or racemic absolute configuration.

In one embodiment, the radical -[AA] is an amino acid residue in theform of a racemic mixture.

In one embodiment, the radical -[AA] is an amino acid residue in theform of isolated isomers of D absolute configuration.

In one embodiment, the radical -[AA] is an amino acid residue in theform of isolated isomers of L absolute configuration.

In one embodiment, u is between 1 and 5.

In one embodiment, u is between 3 and 5.

In one embodiment, u=8.

In one embodiment, u=7.

In one embodiment, u=6.

In one embodiment, u=5.

In one embodiment, u=4.

In one embodiment, u=3.

In one embodiment, u=2.

In one embodiment, u=1.

In one embodiment, hexoses are chosen from the group consisting ofmannose, glucose, fructose, sorbose, tagatose, psicose, galactose,allose, altrose, talose, idose, gulose, fucose, fuculose, rhamnose,mannitol, sorbitol and galactitol (dulcitol).

In one embodiment, the glycoside bonds are of (1,4) or (1,6) type.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,1) type.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of different saccharide units chosen from hexosesand linked via a glycoside bond of (1,1) type, said saccharide backbonebeing chosen from the group consisting of trehalose and sucrose.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,2) type.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,2) type, said saccharidebackbone being kojibiose.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,3) type.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,3) type, said saccharidebackbone being chosen from the group consisting of nigeriose andlaminaribiose.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,4) type.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,4) type, said saccharidebackbone being chosen from the group consisting of maltose, lactose andcellobiose.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,6) type.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,6) type, said saccharidebackbone being chosen from the group consisting of isomaltose, melibioseand gentiobiose.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of identical or different saccharide units chosenfrom hexoses linked via a glycoside bond of (1,6) type, said saccharidebackbone being isomaltose.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of saccharide units, one of which is in cyclic formand the other in open reduced form.

In one embodiment, the substituted anionic compound is chosen fromanionic compounds consisting of a saccharide backbone formed from adiscrete number u=2 of saccharide units, one of which is in cyclic formand the other in open reduced form, said saccharide backbone beingchosen from the group consisting of maltitol and isomaltitol.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is formedfrom a discrete number 3≤u≤8 of identical or different saccharide units.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that at least one of the identical ordifferent saccharide units, of which the saccharide backbone formed froma discrete number 3≤u≤8 of saccharide units is composed, is chosen fromthe group consisting of hexose units linked via identical or differentglycoside bonds.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits, of which the saccharide backbone formed from a discrete number3≤u≤8 of saccharide units is composed, are chosen from hexoses andlinked via at least one glycoside bond of (1,2) type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits, of which the saccharide backbone formed from a discrete number3≤u≤8 of saccharide units is composed, are chosen from hexoses andlinked via at least one glycoside bond of (1,3) type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits, of which the saccharide backbone formed from a discrete number3≤u≤8 of saccharide units is composed, are chosen from hexoses andlinked via at least one glycoside bond of (1,4) type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits, of which the saccharide backbone formed from a discrete number3≤u≤8 of saccharide units is composed, are chosen from hexoses andlinked via at least one glycoside bond of (1,6) type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is formedfrom a discrete number u=3 of identical or different saccharide units.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that it comprises at least one saccharideunit chosen from the group consisting of hexoses in cyclic form and atleast one saccharide unit chosen from the group consisting of hexoses inopen form.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the three saccharide units areidentical.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that two of the three saccharide units areidentical.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical saccharide units arechosen from hexoses, two of which are in cyclic form and one is in openreduced form, and linked via glycoside bonds of (1,4) type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical saccharide units arechosen from hexoses, two of which are in cyclic form and one is in openreduced form, and linked via glycoside bonds of (1,6) type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and in that the central hexose is linkedvia a glycoside bond of (1,2) type and via a glycoside bond of (1,4)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and in that the central hexose is linkedvia a glycoside bond of (1,3) type and via a glycoside bond of (1,4)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and in that the central hexose is linkedvia a glycoside bond of (1,2) type and via a glycoside bond of (1,6)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and in that the central hexose is linkedvia a glycoside bond of (1,2) type and via a glycoside bond of (1,3)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and in that the central hexose is linkedvia a glycoside bond of (1,4) type and via a glycoside bond of (1,6)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is erlose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the three identical or differentsaccharide units are hexose units chosen from the group consisting ofmannose and glucose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone ismaltotriose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone isisomaltotriose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is formedfrom a discrete number u=4 of identical or different saccharide units.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the four saccharide units areidentical.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that three of the four saccharide unitsare identical.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the four saccharide units are hexoseunits chosen from the group consisting of mannose and glucose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone ismaltotetraose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and in that a terminal hexose is linkedvia a glycoside bond of (1,2) type and in that the others are linkedtogether via a glycoside bond of (1,6) type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and linked via a glycoside bond of (1,6)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is formedfrom a discrete number u=5 of identical or different saccharide units.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the five saccharide units areidentical.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the five saccharide units are hexoseunits chosen from the group consisting of mannose and glucose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and linked via a glycoside bond of (1,4)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone ismaltopentaose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is formedfrom a discrete number u=6 of identical or different saccharide units.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the six saccharide units areidentical.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and linked via a glycoside bond of (1,4)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the six identical or differentsaccharide units are hexose units chosen from the group consisting ofmannose and glucose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone ismaltohexaose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is formedfrom a discrete number u=7 of identical or different saccharide units.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the seven saccharide units areidentical.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and linked via a glycoside bond of (1,4)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the seven saccharide units are hexoseunits chosen from the group consisting of mannose and glucose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone ismaltoheptaose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is formedfrom a discrete number u=8 of identical or different saccharide units.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the eight saccharide units areidentical.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the identical or different saccharideunits are chosen from hexoses and linked via a glycoside bond of (1,4)type.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the eight saccharide units are hexoseunits chosen from the group consisting of mannose and glucose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone ismaltooctaose.

In one embodiment, the substituted anionic compound comprising adiscrete number of saccharide units is a natural compound.

In one embodiment, the substituted anionic compound comprising adiscrete number of saccharide units is a synthetic compound.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that it is obtained by enzymaticdegradation of a polysaccharide followed by purification.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that it is obtained by chemicaldegradation of a polysaccharide followed by purification.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that it is obtained chemically, bycovalent coupling of precursors of lower molecular weight.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is sophorose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is sucrose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is lactulose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is maltulose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is leucrose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is rutinose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone isisomaltulose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone isfucosyllactose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone isgentianose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is raffinose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone ismelezitose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is panose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is kestose.

In one embodiment, the substituted anionic compound according to theinvention is characterized in that the saccharide backbone is stachyose.

In one embodiment, the polyanionic compound is a non-polymericpolyanionic (NPP) compound whose affinity for zinc is less than theaffinity of insulin for zinc and whose dissociation constantKd_(Ca)=[NPP compound]^(r) [Ca²⁺]^(s)/[(NPP compound)^(r)-(Ca²⁺)^(s)] isless than or equal to 10^(−1.5).

The dissociation constants (Kd) of the various polyanionic compoundswith respect to calcium ions are determined by external calibrationusing an electrode specific for calcium ions (Mettler-Toledo) and areference electrode. All the measurements are performed in 150 mM NaClat pH 7. Only the concentrations of free calcium ions are determined;the calcium ions bound to the polyanionic compound do not induce anyelectrode potential.

In one embodiment, the polyanionic compound is chosen from the groupconsisting of polycarboxylic acids and the Na⁺, K⁺, Ca²⁺ or Mg²⁺ saltsthereof.

In one embodiment, the polycarboxylic acid is chosen from the groupconsisting of citric acid and tartaric acid, and the Na⁺, K⁺, Ca²⁺ orMg²⁺ salts thereof.

In one embodiment, the polyanionic compound is chosen from the groupconsisting of polyphosphoric acids and the Na⁺, K⁺, Ca²⁺ or Mg²⁺ saltsthereof.

In one embodiment, the polyphosphoric acid is triphosphate and the Na⁺,K⁺, Ca²⁺ or Mg²⁺ salts thereof.

In one embodiment, the polyanionic compound is citric acid and the Na⁺,K⁺, Ca²⁺ or Mg²⁺ salts thereof.

In one embodiment, the polyanionic compound is tartaric acid and theNa⁺, K⁺, Ca²⁺ or Mg²⁺ salts thereof.

In one embodiment, the polyanionic compound is triphosphoric acid andthe Na⁺, K⁺, Ca²⁺ or Mg²⁺ salts thereof.

In one embodiment, the polyanionic compound is a compound consisting ofa saccharide backbone formed from a discrete number of saccharide unitsobtained from a disaccharide compound chosen from the group consistingof trehalose, maltose, lactose, sucrose, cellobiose, isomaltose,maltitol and isomaltitol.

In one embodiment, the polyanionic compound consisting of a saccharidebackbone formed from a discrete number of saccharide units is obtainedfrom a compound consisting of a backbone formed from a discrete numberof saccharide units chosen from the group consisting of maltotriose,maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaoseand isomaltotriose.

In one embodiment, the polyanionic compound consisting of a saccharidebackbone formed from a discrete number of saccharide units is chosenfrom the group consisting of carboxymethylmaltotriose,carboxymethylmaltotetraose, carboxymethylmaltopentaose,carboxymethylmaltohexaose, carboxymethylmaltoheptaose,carboxymethylmaltooctaose and carboxymethylisomaltotriose.

In one embodiment, the ratio (number of moles of acid functions borne bythe polyanionic compound/number of moles of anionic compound) is greaterthan or equal to 3.

In one embodiment, the ratio (number of moles of acid functions borne bythe polyanionic compound/number of moles of anionic compound) is greaterthan or equal to 4.

In one embodiment, the ratio (number of moles of acid functions borne bythe polyanionic compound consisting of a saccharide backbone/number ofmoles of anionic compound) is greater than or equal to 5.

In one embodiment, the ratio (number of moles of acid functions borne bythe polyanionic compound consisting of a saccharide backbone/number ofmoles of anionic compound) is greater than or equal to 8.

In one embodiment, the substituted anionic compound/insulin mole ratiosare between 0.6 and 75.

In one embodiment, the mole ratios are between 0.7 and 50.

In one embodiment, the mole ratios are between 1.4 and 35.

In one embodiment, the mole ratios are between 1.9 and 30.

In one embodiment, the mole ratios are between 2.3 and 30.

In one embodiment, the substituted anionic compound/insulin mole ratiois equal to 8.

In one embodiment, the substituted anionic compound/insulin mole ratiois equal to 12.

In one embodiment, the substituted anionic compound/insulin mole ratiois equal to 16.

In one embodiment, the substituted anionic compound/insulin mass ratiosare between 0.5 and 10.

In one embodiment, the mass ratios are between 0.6 and 7.

In one embodiment, the mass ratios are between 1.2 and 5.

In one embodiment, the mass ratios are between 1.6 and 4.

In one embodiment, the mass ratios are between 2 and 4.

In one embodiment, the substituted anionic compound/insulin mass ratiois 2.

In one embodiment, the substituted anionic compound/insulin mass ratiois 3.

In one embodiment, the substituted anionic compound/insulin mass ratiois 4.

In one embodiment, the substituted anionic compound/insulin mass ratiois 6.

In one embodiment, the concentration of substituted anionic compound isbetween 1.8 and 36 mg/mL.

In one embodiment, the concentration of substituted anionic compound isbetween 1.8 and 36.5 mg/mL.

In one embodiment, the concentration of substituted anionic compound isbetween 2.1 and 25 mg/mL.

In one embodiment, the concentration of substituted anionic compound isbetween 4.2 and 18 mg/mL.

In one embodiment, the concentration of substituted anionic compound isbetween 5.6 and 15 mg/mL.

In one embodiment, the concentration of substituted anionic compound isbetween 7 and 15 mg/mL.

In one embodiment, the concentration of substituted anionic compound is7.3 mg/mL.

In one embodiment, the concentration of substituted anionic compound is10.5 mg/mL.

In one embodiment, the concentration of substituted anionic compound is14.6 mg/mL.

In one embodiment, the concentration of substituted anionic compound is21.9 mg/mL.

In one embodiment, the concentration of polyanionic compound is between2 and 150 mM.

In one embodiment, the concentration of polyanionic compound is between2 and 100 mM.

In one embodiment, the concentration of polyanionic compound is between2 and 75 mM.

In one embodiment, the concentration of polyanionic compound is between2 and 50 mM.

In one embodiment, the concentration of polyanionic compound is between2 and 30 mM.

In one embodiment, the concentration of polyanionic compound is between2 and 20 mM.

In one embodiment, the concentration of polyanionic compound is between2 and 10 mM.

In one embodiment, the concentration of polyanionic compound is between5 and 150 mM.

In one embodiment, the concentration of polyanionic compound is between5 and 100 mM.

In one embodiment, the concentration of polyanionic compound is between5 and 75 mM.

In one embodiment, the concentration of polyanionic compound is between5 and 50 mM.

In one embodiment, the concentration of polyanionic compound is between5 and 30 mM.

In one embodiment, the concentration of polyanionic compound is between5 and 20 mM.

In one embodiment, the concentration of polyanionic compound is between5 and 10 mM.

In one embodiment, the concentration of polyanionic compound is between0.5 and 30 mg/mL.

In one embodiment, the concentration of polyanionic compound is between0.5 and 25 mg/mL.

In one embodiment, the concentration of polyanionic compound is between0.5 and 10 mg/mL.

In one embodiment, the concentration of polyanionic compound is between0.5 and 8 mg/mL.

In one embodiment, the concentration of polyanionic compound is between1 and 30 mg/mL.

In one embodiment, the concentration of polyanionic compound is between1.5 and 25 mg/mL.

In one embodiment, the concentration of polyanionic compound is between2 and 25 mg/mL.

In one embodiment, the concentration of polyanionic compound is between2 and 10 mg/mL.

In one embodiment, the concentration of polyanionic compound is between2 and 8 mg/mL.

In one embodiment, the substituted anionic compound is sodiummaltotriosemethylcarboxylate modified with sodium phenylalaninate, u=3,i=0.65, j=1.0.

In one embodiment, the substituted anionic compound is sodiummaltotriosemethylcarboxylate modified with sodium phenylalaninate, u=3,i=1.0, j=0.65.

In one embodiment, the substituted anionic compound is sodiummaltotriosemethylcarboxylate modified with sodium phenylalaninate, u=3,i=0.46, j=1.2.

In one embodiment, the substituted anionic compound is sodiummaltotriosemethylcarboxylate modified with sodium phenylalaninate, u=3,i=0.35, j=0.65.

In one embodiment, the polyanionic compound is sodiummaltotriosemethylcarboxylate.

In one embodiment, the polyanionic compound is sodium citrate.

In one embodiment, the polyanionic compound is triphosphate in acidicform or in basic form in the form of the sodium salt or the potassiumsalt.

In one embodiment, the polyanionic compound is tartrate in acidic formor in basic form in the form of the sodium salt or the potassium salt.

The invention also relates to an insulin pharmaceutical formulationcomprising a composition according to the invention, in which theinsulin is in hexameric form.

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is between 240 and 3000μM (40 to 500 IU/mL).

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is between 600 and 3000μM (100 to 500 IU/mL).

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is between 600 and 2400μM (100 to 400 IU/mL).

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is between 600 and 1800μM (100 to 300 IU/mL).

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is between 600 and 1200μM (100 to 200 IU/mL).

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is 600 μM (100 IU/mL).

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is 1200 μM (200 IU/mL).

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is 1800 μM (300 IU/mL).

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is 2400 μM (400 IU/mL).

In one embodiment, it relates to a pharmaceutical formulationcharacterized in that the insulin concentration is 3000 μM (500 IU/mL).

The invention relates to the use of at least one substituted anioniccompound, said compound consisting of a saccharide backbone formed froma discrete number u of between 1 and 8 (1≤u≤8) of identical or differentsaccharide units, linked via identical or different glycoside bonds,said saccharide units being chosen from the group consisting of hexoses,in cyclic form or in open reduced form, said compound comprisingpartially substituted carboxyl functional groups, the unsubstitutedcarboxyl functional groups being salifiable to prepare a pharmaceuticalformulation of human insulin, in combination with a polyanioniccompound, making it possible, after administration, to accelerate thepassage of the insulin into the blood and to reduce glycemia morerapidly when compared with a formulation free of substituted anioniccompound, and optionally of anionic compounds.

In one embodiment, the invention relates to the use of at least onesubstituted anionic compound, said compound comprising partiallysubstituted carboxyl functional groups, the unsubstituted carboxylfunctional groups being salifiable, to prepare a pharmaceuticalformulation of human insulin, in combination with a polyanioniccompound, making it possible, after administration, to accelerate thepassage of the human insulin into the blood and to reduce glycemia morerapidly when compared with a formulation free of substituted anioniccompound, and optionally of anionic compounds.

In one embodiment, the invention relates to the use of at least onesubstituted anionic compound, said compound comprising partiallysubstituted carboxyl functional groups, the unsubstituted carboxylfunctional groups being salifiable, to prepare an insulin analogformulation, in combination with a polyanionic compound, making itpossible, after administration, to accelerate the passage of the insulinanalog into the blood and to reduce glycemia more rapidly when comparedwith a formulation free of substituted anionic compound, and optionallyof anionic compounds.

In one embodiment, the insulin is human insulin.

The term “human insulin” means an insulin obtained by synthesis orrecombination, whose peptide sequence is the sequence of human insulin,including the allelic variations and homologs.

In one embodiment, the insulin is a recombinant human insulin asdescribed in the European Pharmacopea and the American Pharmacopea.

In one embodiment, the insulin is an insulin analog.

The term “insulin analog” means a recombinant insulin whose primarysequence contains at least one modification relative to the primarysequence of human insulin.

In one embodiment, the insulin analog is chosen from the groupconsisting of the insulin lispro (Humalog®), the insulin aspart(Novolog®, Novorapid®) and the insulin glulisine (Apidra®).

In one embodiment, the insulin analog is the insulin lispro (Humalog®).

In one embodiment, the insulin analog is the insulin aspart (Novolog®,Novorapid®).

In one embodiment, the insulin analog is the insulin glulisine(Apidra®).

In one embodiment, the use is characterized in that the substitutedanionic compound is chosen from substituted anionic compounds, inisolated form or as a mixture, consisting of a saccharide backboneformed from a discrete number u of between 1 and 8 (1≤u≤8) of identicalor different saccharide units, linked via identical or differentglycoside bonds, said saccharide units being chosen from hexoses, incyclic form or in open reduced form, characterized in that they aresubstituted with:

-   -   a) at least one substituent of general formula I:        —[R₁]_(a)-[AA]_(m)  Formula I        -   the substituents being identical or different when there are            at least two substituents, in which:        -   the radical -[AA] denotes an amino acid residue,        -   the radical —R₁— being:            -   either a bond and then a=0 and the amino acid residue                -[AA]- is directly linked to the backbone via a function                G,            -   or a C2 to C15 carbon-based chain, and then a=1,                optionally substituted and/or comprising at least one                heteroatom chosen from O, N and S and at least one acid                function before the reaction with the amino acid, said                chain forming with the amino acid residue -[AA]- an                amide function, and is attached to the backbone by means                of a function F resulting from a reaction between a                hydroxyl function borne by the backbone and a function                or substituent borne by the precursor of the radical                —R₁—,        -   F is a function chosen from ether, ester and carbamate            functions,        -   G is a carbamate function,        -   m is equal to 1 or 2,        -   the degree of substitution of the saccharide units, j, in            —[R₁]_(a)-[AA]_(m) being strictly greater than 0 and less            than or equal to 6, 0<j≤6    -   b) and, optionally, one or more substituents —R′₁,        -   the substituent —R′₁ being a C2 to C15 carbon-based chain,            which is optionally substituted and/or comprising at least            one heteroatom chosen from O, N and S and at least one acid            function in the form of an alkali metal cation salt, said            chain being linked to the backbone via a function F′            resulting from a reaction between a hydroxyl function borne            by the backbone and a function or substituent borne by the            precursor of the substituent —R′₁,        -   F′ is an ether, ester or carbamate function,        -   the degree of substitution of the saccharide units, i, in            —R′₁, being between 0 and 6−j, 0≤i≤6−j, and        -   F and F′ are identical or different,        -   F and G are identical or different,            -   i+j≤6,        -   —R′₁ is identical to or different from —R₁—,        -   the free salifiable acid functions borne by the substituent            —R′₁ are in the form of alkali metal cation salts,        -   said glycoside bonds, which may be identical or different,            being chosen from the group consisting of glycoside bonds of            (1,1), (1,2), (1,3), (1,4) or (1,6) type, in an alpha or            beta geometry.

In one embodiment, the use is characterized in that the substitutedanionic compound, in isolated form or as a mixture, is chosen fromsubstituted anionic compounds consisting of a saccharide backbone formedfrom a discrete number u of between 1 and 8 (1≤u≤8) of identical ordifferent saccharide units, linked via identical or different glycosidebonds, said saccharide units being chosen from hexoses, in cyclic formor in open reduced form, characterized

-   -   a) in that they are randomly substituted with:        -   at least one substituent of general formula I:            —[R₁]_(a)-[AA]_(m)  Formula I    -   the substituents being identical or different when there are at        least two substituents, in which:    -   the radical -[AA]- denotes an amino acid residue, said amino        acid being chosen from the group consisting of phenylalanine,        alpha-methylphenylalanine, 3,4-dihydroxyphenylalanine, tyrosine,        alpha-methyltyrosine, O-methyltyrosine, alpha-phenylglycine,        4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine, and the        alkali metal cation salts thereof, said derivatives being in L        or D absolute configuration, -[AA]- is attached to the backbone        of the molecule via a linker arm —R₁— or directly attached to        the backbone via a function G,    -   —R₁— being:        -   either a bond G, and then a=0,        -   or a C2 to C15 carbon-based chain, and then a=1, optionally            substituted and/or comprising at least one heteroatom chosen            from O, N and S and bearing at least one acid function            before the reaction with the amino acid, said chain forming            with the amino acid residue -[AA]- an amide bond, and is            attached to the saccharide backbone by means of a function F            resulting from a reaction between a hydroxyl function borne            by the backbone and a function borne by the precursor of R₁,    -   F is an ether, ester or carbamate function,    -   G is a carbamate function,    -   m is equal to 1 or 2,    -   the degree of substitution, j, in —[R₁]_(a)[AA]_(m) being        strictly greater than 0 and less than or equal to 6, 0<j≤6,    -   and, optionally,        -   one or more substituents —R′₁    -   —R′₁ being a C2 to C15 carbon-based chain, which is optionally        substituted and/or comprising at least one heteroatom (such as        O, N and S) and bearing at least one acid function in the form        of an alkali metal cation salt, said chain being attached to the        saccharide backbone via a function F′ resulting from a reaction        between a hydroxyl function borne by the backbone and a function        borne by the precursor of —R′₁,    -   F′ is an ether, ester or carbamate function,    -   the degree of substitution, i, in —R′₁, being between 0 and 6−j,        0≤i≤6−j, and    -   —R′₁— is identical to or different from —R₁,    -   F and F′ are identical or different,    -   F′ and G are identical or different,    -   the free salifiable acid functions are in the form of alkali        metal cation salts,    -   b) said glycoside bonds, which may be identical or different,        being chosen from the group consisting of glycoside bonds of        (1,1), (1,2), (1,3), (1,4) or (1,6) type, in an alpha or beta        geometry,    -   c) i+j≤6.

In one embodiment, m is equal to 1.

In one embodiment, —R1 and —R′1, which may be identical or different,are a C1 to C8 carbon-based chain.

In one embodiment, —R1 and —R′1, which may be identical or different,are a C1 to C4 carbon-based chain.

In one embodiment, —R1 and —R′1, which may be identical or different,are a C1 to C2 carbon-based chain.

It is known to those skilled in the art that the delay of action ofinsulins is dependent on the insulin concentration. Only the delay ofaction values for formulations at 100 IU/mL are documented.

“Regular” human insulin formulations on the market at a concentration of600 μM (100 IU/mL) have a delay of action of between 50 and 90 minutesand an end of action of about 360 to 420 minutes in man. The time toachieve the maximum insulin concentration in the blood is between 90 and180 minutes in man.

Rapid insulin analog formulations on the market at a concentration of600 μM (100 IU/mL) have a delay of action of between 30 and 60 minutesand an end of action of about 240-300 minutes in man. The time toachieve the maximum insulin concentration in the blood is between 50 and90 minutes in man.

The invention also relates to a method for preparing a human insulinformulation having an insulin concentration of between 240 and 3000 μM(40 and 500 IU/mL), whose delay of action in man is less than that ofthe reference formulation at the same insulin concentration in theabsence of a substituted anionic compound and of a polyanionic compound,characterized in that it comprises (1) a step of adding to saidformulation at least one substituted anionic compound, said compoundcomprising partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable, and (2) astep of adding to said formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing a human insulinformulation having an insulin concentration of between 600 and 1200 μM(100 and 200 IU/mL), whose delay of action in man is less than that ofthe reference formulation at the same insulin concentration in theabsence of a substituted anionic compound and of a polyanionic compound,characterized in that it comprises (1) a step of adding to saidformulation at least one substituted anionic compound, said compoundcomprising partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable, and (2) astep of adding to said formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing a human insulinformulation having an insulin concentration of 600 μM (100 IU/mL), whosedelay of action in man is less than 60 minutes, characterized in that itcomprises (1) a step of adding to said formulation at least onesubstituted anionic compound, said compound comprising partiallysubstituted carboxyl functional groups, the unsubstituted carboxylfunctional groups being salifiable, and (2) a step of adding to saidformulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing a human insulinformulation having an insulin concentration of 1200 μM (200 IU/mL),whose delay of action in man is at least 10% less than that of the humaninsulin formulation at the same concentration (200 IU/mL) and in theabsence of a substituted anionic compound and of a polyanionic compound,characterized in that it comprises (1) a step of adding to saidformulation at least one substituted anionic compound, said compoundcomprising partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable, and (2) astep of adding to said formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing a human insulinformulation having an insulin concentration of 1800 μM (300 IU/mL),whose delay of action in man is at least 10% less than that of the humaninsulin formulation at the same concentration (300 IU/mL) and in theabsence of a substituted anionic compound and of a polyanionic compound,characterized in that it comprises (1) a step of adding to saidformulation at least one substituted anionic compound, said compoundcomprising partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable, and (2) astep of adding to said formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing a human insulinformulation having an insulin concentration of 2400 μM (400 IU/mL),whose delay of action in man is at least 10% less than that of the humaninsulin formulation at the same concentration (400 IU/mL) and in theabsence of a substituted anionic compound and of a polyanionic compound,characterized in that it comprises (1) a step of adding to saidformulation at least one substituted anionic compound, said compoundcomprising partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable, and (2) astep of adding to said formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing a human insulinformulation having an insulin concentration of 3000 μM (500 IU/mL),whose delay of action in man is at least 10% less than that of the humaninsulin formulation at the same concentration (500 IU/mL) and in theabsence of a substituted anionic compound and of a polyanionic compound,characterized in that it comprises (1) a step of adding to saidformulation at least one substituted anionic compound, said compoundcomprising partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable, and (2) astep of adding to said formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention consists in preparing a rapid human insulin formulation,characterized in that it comprises (1) a step of adding to saidformulation at least one substituted anionic compound, said compoundcomprising partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable, and (2) astep of adding to said formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing a human insulinformulation at a concentration of 600 μM (100 IU/mL), whose delay ofaction in man is less than 60 minutes, preferably less than 45 minutesand more preferably less than 30 minutes, characterized in that itcomprises (1) a step of adding to said formulation at least onesubstituted anionic compound, said compound comprising partiallysubstituted carboxyl functional groups, the unsubstituted carboxylfunctional groups being salifiable, and (2) a step of adding to saidformulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing an insulin analogformulation having an insulin concentration of between 240 and 3000 μM(40 and 500 IU/mL), whose delay of action in man is less than that ofthe reference formulation at the same insulin concentration in theabsence of a substituted anionic compound and of a polyanionic compound,characterized in that it comprises (1) a step of adding to saidformulation at least one substituted anionic compound, said compoundcomprising partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable, and (2) astep of adding to said formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing an insulin analogformulation having an insulin concentration of between 600 and 1200 μM(100 and 200 IU/mL), whose delay of action in man is less than that ofthe reference formulation at the same insulin analog concentration inthe absence of a substituted anionic compound and of a polyanioniccompound, characterized in that it comprises (1) a step of adding tosaid formulation at least one substituted anionic compound, saidcompound comprising partially substituted carboxyl functional groups,the unsubstituted carboxyl functional groups being salifiable, and (2) astep of adding to said formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing an insulin analogformulation having an insulin concentration of 600 μmol/L (100 IU/mL),whose delay of action in man is less than 30 minutes, characterized inthat it comprises (1) a step of adding to said formulation at least onesubstituted anionic compound, said compound comprising partiallysubstituted carboxyl functional groups, the unsubstituted carboxylfunctional groups being salifiable, and (2) a step of adding to saidformulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing an insulin analogformulation having an insulin concentration of 1200 μM (200 IU/mL),whose delay of action in man is at least 10% less than that of theinsulin analog formulation in the absence of a substituted anioniccompound and of a polyanionic compound, characterized in that itcomprises (1) a step of adding to said formulation at least onesubstituted anionic compound, said compound comprising partiallysubstituted carboxyl functional groups, the unsubstituted carboxylfunctional groups being salifiable, and (2) a step of adding to saidformulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing an insulin analogformulation having an insulin concentration of 1800 μM (300 IU/mL),whose delay of action in man is at least 10% less than that of theinsulin analog formulation in the absence of a substituted anioniccompound and of a polyanionic compound, characterized in that itcomprises (1) a step of adding to said formulation at least onesubstituted anionic compound, said compound comprising partiallysubstituted carboxyl functional groups, the unsubstituted carboxylfunctional groups being salifiable, and (2) a step of adding to saidformulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing an insulin analogformulation having an insulin concentration of 2400 μM (400 IU/mL),whose delay of action in man is at least 10% less than that of theinsulin analog formulation in the absence of a substituted anioniccompound and of a polyanionic compound, characterized in that itcomprises (1) a step of adding to said formulation at least onesubstituted anionic compound, said compound comprising partiallysubstituted carboxyl functional groups, the unsubstituted carboxylfunctional groups being salifiable, and (2) a step of adding to saidformulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention also relates to a method for preparing an insulin analogformulation having an insulin concentration of 3000 μM (500 IU/mL),whose delay of action in man is at least 10% less than that of theinsulin analog formulation in the absence of a substituted anioniccompound and of a polyanionic compound, characterized in that itcomprises (1) a step of adding to said formulation at least onesubstituted anionic compound, said compound comprising partiallysubstituted carboxyl functional groups, the unsubstituted carboxylfunctional groups being salifiable, and (2) a step of adding to saidformulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

The invention consists in preparing a very rapid insulin analogformulation, characterized in that it comprises a step of adding to saidformulation at least one substituted anionic compound, said compoundcomprising partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable.

In one embodiment, the preparation also comprises a step of adding tosaid formulation at least one polyanionic compound.

In one embodiment, the insulin is in hexameric form.

In one embodiment, the insulin analog is chosen from the groupconsisting of the insulin lispro (Humalog®), the insulin aspart(Novolog®, Novorapid®) and the insulin glulisine (Apidra®).

In one embodiment, the insulin analog is the insulin lispro (Humalog®).

In one embodiment, the insulin analog is the insulin aspart (Novolog®,Novorapid®).

In one embodiment, the insulin analog is the insulin glulisine(Apidra®).

In one embodiment, the insulin is a recombinant human insulin asdescribed in the European Pharmacopea and the American Pharmacopea.

In one embodiment, the insulin is an insulin analog chosen from thegroup consisting of the insulin lispro (Humalog®), the insulin aspart(Novolog®, Novorapid®) and the insulin glulisine (Apidra®).

The composition may furthermore be prepared by simple mixing of anaqueous solution of human insulin or of insulin analog and an aqueoussolution of substituted anionic compound as a mixture with a polyanioniccompound.

In one embodiment, the composition may be prepared by simple mixing ofan aqueous solution of human insulin or of insulin analog, an aqueoussolution of substituted anionic compound and a polyanionic compound insolution or in lyophilizate form.

In one embodiment, the composition may be prepared by simple mixing ofan aqueous solution of human insulin or of insulin analog, a substitutedanionic compound in lyophilizate form and a polyanionic compound insolution or in lyophilizate form.

Preferably, this composition is in the form of an injectable solution.

In one embodiment, the concentration of human insulin or insulin analogis between 240 and 3000 μM (40 to 500 IU/mL).

In one embodiment, the concentration of human insulin or insulin analogis between 600 and 3000 μM (100 to 500 IU/mL).

In one embodiment, the concentration of human insulin or insulin analogis between 600 and 2400 μM (100 to 400 IU/mL).

In one embodiment, the concentration of human insulin or insulin analogis between 600 and 1800 μM (100 to 300 IU/mL).

In one embodiment, the concentration of human insulin or insulin analogis between 600 and 1200 μM (100 to 200 IU/mL).

In one embodiment, the concentration of human insulin or insulin analogis 600 μM (100 IU/mL).

In one embodiment, the concentration of human insulin or insulin analogis 1200 μM (200 IU/mL).

In one embodiment, the concentration of human insulin or insulin analogof 600 μM (100 IU/mL) may be reduced by simple dilution, in particularfor pediatric applications.

In one embodiment, the concentration of human insulin or insulin analogis 1800 μM (300 IU/mL).

In one embodiment, the concentration of human insulin or insulin analogis 2400 μM (400 IU/mL).

In one embodiment, the concentration of human insulin or insulin analogis 3000 μM (500 IU/mL).

The invention also relates to a pharmaceutical formulation according tothe invention, characterized in that it is obtained by drying and/orlyophilization.

In one embodiment, the compositions according to the invention alsocomprise the addition of zinc salts to a concentration of between 0 and500 μM.

In one embodiment, the compositions according to the invention alsocomprise the addition of zinc salts to a concentration of between 0 and300 μM.

In one embodiment, the compositions according to the invention alsocomprise the addition of zinc salts to a concentration of between 0 and200 μM.

In one embodiment, the compositions according to the invention comprisebuffers at concentrations of between 0 and 100 mM, preferably between 0and 50 mM or between 15 and 50 mM.

In one embodiment, the buffer is Tris.

In one embodiment, the compositions according to the invention alsocomprise preservatives.

In one embodiment, the preservatives are chosen from the groupconsisting of m-cresol and phenol, alone or as a mixture.

In one embodiment, the concentration of preservatives is between 10 and50 mM.

In one embodiment, the concentration of preservatives is between 10 and40 mM.

The compositions according to the invention may also comprise additivessuch as tonicity agents, for instance glycerol, sodium chloride (NaCl),mannitol and glycine.

The compositions according to the invention may also comprise additivesin accordance with the pharmacopeas, for instance surfactants, forexample polysorbate.

The compositions according to the invention may also comprise anyexcipient in accordance with the pharmacopeas which are compatible withthe insulins used at the working concentrations.

In the case of local and systemic release, the envisaged modes ofadministration are intravenous, subcutaneous, intradermal orintramuscular.

Transdermal, oral, nasal, vaginal, ocular, oral and pulmonaryadministration routes are also envisaged.

The invention also relates to the use of a composition according to theinvention for the formulation of a solution of human insulin or insulinanalog in a concentration of 100 IU/mL intended for implantable ortransportable insulin pumps.

The invention also relates to the use of a composition according to theinvention for the formulation of a solution of human insulin or insulinanalog in a concentration of 200 IU/mL intended for implantable ortransportable insulin pumps.

The invention also relates to substituted anionic compound, in isolatedform or as a mixture, chosen from substituted anionic compoundsconsisting of a saccharide backbone formed from a discrete number u ofbetween 1 and 8 (1≤u≤8) of identical or different saccharide units,linked via identical or different glycoside bonds, said saccharide unitsbeing chosen from hexoses, in cyclic form or in open reduced form,characterized in that they are substituted with:

-   -   a) at least one substituent of general formula I:        —[R₁]_(a)-[AA]_(m)  Formula I        -   the substituents being identical or different when there are            at least two substituents, in which:        -   the radical -[AA] denotes an amino acid residue,        -   the radical —R₁— being:            -   either a bond and then a=0 and the amino acid residue                -[AA]- is directly linked to the backbone via a function                G,            -   or a C2 to C15 carbon-based chain, and then a=1,                optionally substituted and/or comprising at least one                heteroatom chosen from O, N and S and at least one acid                function before the reaction with the amino acid, said                chain forming with the amino acid residue -[AA]- an                amide function, and is attached to the backbone by means                of a function F resulting from a reaction between a                hydroxyl function borne by the backbone and a function                or substituent borne by the precursor of the radical                —R₁—,        -   F is a function chosen from ether, ester and carbamate            functions,        -   G is a carbamate function,        -   m is equal to 1 or 2,        -   the degree of substitution of the saccharide units, j, in            —[R₁]_(a)-[AA]_(m) being strictly greater than 0 and less            than or equal to 6, 0<j≤6    -   b) and, optionally, one or more substituents —R′₁,        -   the substituent —R′₁ being a C2 to C15 carbon-based chain,            which is optionally substituted and/or comprising at least            one heteroatom chosen from O, N and S and at least one acid            function in the form of an alkali metal cation salt, said            chain being linked to the backbone via a function F′            resulting from a reaction between a hydroxyl function borne            by the backbone and a function or substituent borne by the            precursor of the substituent —R′₁,        -   F′ is an ether, ester or carbamate function,        -   the degree of substitution of the saccharide units, i, in            —R′₁, being between 0 and 6−j, 0≤i≤6−j, and        -   F and F′ are identical or different,        -   F and G are identical or different,            -   i+j≤6,        -   —R′₁ is identical to or different from —R₁—,        -   the free salifiable acid functions borne by the substituent            —R′₁ are in the form of alkali metal cation salts,        -   said glycoside bonds, which may be identical or different,            being chosen from the group consisting of glycoside bonds of            (1,1), (1,2), (1,3), (1,4) or (1,6) type, in an alpha or            beta geometry.

In the above formula, the different variables have the values mentionedabove.

The substituted anionic compounds according to the invention may beobtained by random grafting of substituents onto the saccharidebackbone.

In one embodiment, the substituted anionic compounds chosen from anioniccompounds substituted with substituents of formula I or II arecharacterized in that they may be obtained by grafting substituents inprecise positions onto the saccharide units via a process involvingsteps of protection/deprotection of the alcohol or carboxylic acidgroups naturally borne by the backbone. This strategy leads to selectivegrafting, especially regioselective grafting, of the substituents ontothe backbone. The protecting groups include, without limitation, thosedescribed in the publication (Wuts, P. G. M. et al., Greene's ProtectiveGroups in Organic Synthesis 2007).

The saccharide backbone may be obtained by degradation of a highmolecular weight polysaccharide. The degradation routes include, withoutlimitation, chemical degradation and/or enzymatic degradation.

The saccharide backbone may also be obtained by formation of glycosidebonds between monosaccharide or oligosaccharide molecules using achemical or enzymatic coupling strategy. The coupling strategies includethose described in the publication (Smoot, J. T. et al., Advances inCarbohydrate Chemistry and Biochemistry 2009, 62, 162-250) and in thepublication (Lindhorst, T. K., Essentials of Carbohydrate Chemistry andBiochemistry 2007, 157-208). The coupling reactions may be performed insolution or on a solid support. The saccharide molecules before couplingmay bear substituents of interest and/or may be functionalized oncecoupled together, randomly or regioselectively.

Thus, by way of example, the compounds according to the invention may beobtained according to one of the following processes:

-   -   the random grafting of substituents onto a saccharide backbone    -   one or more steps of glycosylation between monosaccharide or        oligosaccharide molecules bearing substituents    -   one or more steps of glycosylation between one or more        monosaccharide or oligosaccharide molecules bearing substituents        and one or more monosaccharide or oligosaccharide molecules    -   one or more steps of introduction of protecting groups onto        alcohols or acids naturally borne by the saccharide backbone,        followed by one or more substituent grafting reactions and        finally a step of removal of the protecting groups    -   one or more steps of glycosylation between one or more        monosaccharide or oligosaccharide molecules bearing protecting        groups on alcohols or acids naturally borne by the saccharide        backbone, one or more steps of grafting substituents onto the        backbone obtained, and then a step of removal of the protecting        groups    -   one or more steps of glycosylation between one or more        monosaccharide or oligosaccharide molecules bearing protecting        groups on alcohols or acids naturally borne by the saccharide        backbone, and one or more monosaccharide or oligosaccharide        molecules, one or more substituent grafting steps and then a        step of removal of the protecting groups.

The compounds according to the invention, isolated or as a mixture, maybe separated and/or purified in various ways, especially after havingbeen obtained via the processes described above.

Mention may be made in particular of chromatographic methods, especially“preparative” methods such as:

-   -   flash chromatography, especially on silica, and    -   chromatography such as HPLC (high-performance liquid        chromatography), in particular RP-HPLC (reverse-phase HPLC).

Selective precipitation methods may also be used.

The invention is illustrated by the examples that follow.

EXAMPLES

The structures of the substituted anionic compounds according to theinvention are presented in Table 1. The structures of the polysaccharidecounterexamples are presented in Table 2.

TABLE 1 AA Substituted anionic compounds Com- Substituent Substituentpound i j Saccharide chain —R′₁ —[R₁]_(a)—[AA]_(m)  1 0.65 1.0

 2 1.0 0.65

 3 0.46 1.2

 4 0.35 0.65

 5 1.25 0.4

 6 0.8 0.65

 7 2.65 0.65

 8 1.0 0.75

 9 1.0 0.65

10 0.83 0.81

11 1.12 0.52

R = H, R′₁, —[R₁]_(a)—[AA]_(m)

TABLE 2 AB Polysaccharide counterexamples Weight-average Polysaccharidemolar mass Substituent Substituent counterexamples i j Saccharide chain(kg/mol) —R′₁ —[R₁]_(a)—[AA]_(m) AB1 0.6 0.46

10

AB2 1.01 0.64

5

AB3 0.65 0.45

5

AB4 1.01 0.64

10

AB5 0.45 0.65

5

Polysaccharide counterexamples AB1, AB2, AB3, AB4 and AB5: R = H, R′₁,—[R₁]_(a)—[AA]_(m)

AA1. Compound 1: Sodium Maltotriosemethylcarboxylate Functionalized withSodium L-Phenylalaninate

To 8 g (143 mmol of hydroxyl functions) of maltotriose (CarboSynth)dissolved in water at 65° C. is added 0.6 g (16 mmol) of sodiumborohydride. After stirring for 30 minutes, 28 g (238 mmol) of sodiumchloroacetate are added. To this solution are then added dropwise 24 mLof 10 N NaOH (24 mmol), and the mixture is then heated at 65° C. for 90minutes. 16.6 g (143 mmol) of sodium chloroacetate are then added to thereaction medium, along with dropwise addition of 14 mL of 10 N NaOH (14mmol). After heating for 1 hour, the mixture is diluted with water,neutralized with acetic acid and then purified by ultrafiltration on a 1kDa PES membrane against water. The molecule concentration of the finalsolution is determined on the dry extract, and an acid/base assay in a50/50 (V/V) water/acetone mixture is then performed to determine thedegree of substitution with methylcarboxylate.

According to the dry extract: [compound]=32.9 mg/g

According to the acid/base assay, the degree of substitution withmethylcarboxylate is 1.65 per saccharide unit.

The sodium maltotriosemethylcarboxylate solution is acidified on aPurolite resin (anionic) to obtain maltotriosemethylcarboxylic acid,which is then lyophilized for 18 hours.

10 g of maltotriosemethylcarboxylic acid (63 mmol of methylcarboxylicacid functions) are dissolved in DMF and then cooled to 0° C. A mixtureof ethyl phenylalaninate, hydrochloride salt (8.7 g, 38 mmol) in DMF isprepared. 3.8 g of triethylamine (38 mmol) are added to this mixture. Asolution of NMM (6.3 g, 63 mmol) and of EtOCOCl (6.8 g, 63 mmol) is thenadded to the mixture at 0° C. The ethyl phenylalaninate solution is thenadded and the mixture is stirred at 10° C. An aqueous imidazole solutionis added and the mixture is then heated to 30° C. The medium is dilutedwith water and the solution obtained is then purified by ultrafiltrationon a 1 kDa PES membrane against 0.1 N NaOH, 0.9% NaCl and water. Themolecule concentration of the final solution is determined on the dryextract. A sample of solution is lyophilized and analyzed by ¹H NMR inD₂O to determine the degree of substitution with methylcarboxylatesfunctionalized with sodium L-phenylalaninate.

According to the dry extract: [compound 1]=29.4 mg/g

According to the acid/base assay, the degree of substitution with sodiummethylcarboxylates is 0.65 per saccharide unit.

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 1.0per saccharide unit.

AA2. Compound 2: Sodium Maltotriosemethylcarboxylate Functionalized withSodium L-Phenylalaninate

Via a process similar to that used for the preparation of compound 1, asodium maltotriosecarboxylate functionalized with sodiumL-phenylalaninate is obtained. According to the acid/base assay, thedegree of substitution with sodium methylcarboxylates is 1.0 persaccharide unit.

According to the dry extract: [compound 2]=20.2 mg/g

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.65per saccharide unit.

AA3. Compound 3: Sodium Maltotriosemethylcarboxylate Functionalized withSodium L-Phenylalaninate

Via a process similar to that used for the preparation of compound 1, asodium maltotriosecarboxylate functionalized with sodiumL-phenylalaninate is obtained. According to the acid/base assay, thedegree of substitution with sodium methylcarboxylates is 0.46 persaccharide unit.

According to the dry extract: [compound 3]=7.2 mg/g

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 1.2per saccharide unit.

AA4. Compound 4: Sodium Maltotriosemethylcarboxylate Functionalized withSodium L-Phenylalaninate

Via a process similar to that used for the preparation of compound 1, asodium maltotriosecarboxylate functionalized with sodiumL-phenylalaninate is obtained. According to the acid/base assay, thedegree of substitution with sodium methylcarboxylates is 0.35 persaccharide unit.

According to the dry extract: [compound 4]=3.1 mg/g

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.65per saccharide unit.

AA5. Compound 5: Sodium Maltotriosemethylcarboxylate Functionalized withSodium L-Phenylalaninate

Via a process similar to that used for the preparation of compound 1, asodium maltotriosemethylcarboxylate functionalized with sodiumL-phenylalaninate is obtained.

According to the dry extract: [compound 5]=10.9 mg/g

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.40per saccharide unit.

The degree of substitution with sodium methylcarboxylates is 1.25 persaccharide unit.

AA6. Compound 6: Sodium Maltotriosemethylcarboxylate Functionalized withSodium L-Phenylalaninate

To 8 g (143 mmol of hydroxyl functions) of maltotriose (CarboSynth)dissolved in water at 65° C. is added 0.6 g (16 mmol) of sodiumborohydride. After stirring for 30 minutes, 28 g (237 mmol) of sodiumchloroacetate are added. To this solution are then added dropwise 24 mLof 10 N NaOH (240 mmol). After heating at 65° C. for 90 minutes, themixture is diluted with water, neutralized by adding acetic acid andthen purified by ultrafiltration on a 1 kDa PES membrane against water.The compound concentration of the final solution is determined on thedry extract, and an acid/base assay in a 50/50 (V/V) water/acetonemixture is then performed to determine the degree of substitution withsodium methylcarboxylate.

According to the dry extract: [compound]=14.5 mg/g

According to the acid/base assay, the degree of substitution with sodiummethylcarboxylates is 1.45 per saccharide unit.

The sodium maltotriosemethylcarboxylate solution is acidified on aPurolite resin (anionic) to obtain maltotriosemethylcarboxylic acid,which is then lyophilized for 18 hours.

Via a process similar to that used for the preparation of compound 1, asodium maltotriosemethylcarboxylate functionalized with sodiumL-phenylalaninate is obtained.

According to the dry extract: [compound 6]=10.8 mg/g

According to the ¹H NMR, the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.65per saccharide unit.

The degree of substitution with sodium methylcarboxylates is 0.8 persaccharide unit.

AA7. Compound 7: Sodium Maltotriosemethylcarboxylate Functionalized withSodium L-Phenylalaninate

Via a process similar to that described in the preparation of compound1, 8 g of sodium maltotriosemethylcarboxylate characterized by a degreeof substitution with sodium methylcarboxylate of 1.76 are synthesizedand lyophilized.

8 g (58 mmol of hydroxyl functions) of the lyophilizate and 15 g (129mmol) of sodium chloroacetate are dissolved in water at 65° C. To thissolution are added dropwise 13 mL of 10 N NaOH (130 mmol) and themixture is then heated at 65° C. for 90 minutes. 9 g (78 mmol) of sodiumchloroacetate are then added to the reaction medium, along with dropwiseaddition of 8 mL of 10 N NaOH (80 mmol). After heating for 1 hour, themixture is diluted with water, neutralized with acetic acid and thenpurified by ultrafiltration on a 1 kDa PES membrane against water. Thecompound concentration of the final solution is determined on the dryextract, and an acid/base assay in a 50/50 (V/V) water/acetone mixtureis then performed to determine the degree of substitution with sodiummethylcarboxylates.

According to the dry extract: [compound]=11.7 mg/g

According to the acid/base assay, the degree of substitution with sodiummethylcarboxylates is 3.30 per saccharide unit.

The sodium maltotriosemethylcarboxylate solution is acidified on aPurolite resin (anionic) to obtain maltotriosemethylcarboxylic acid,which is then lyophilized for 18 hours.

Via a process similar to that used for the preparation of compound 1, asodium maltotriosemethylcarboxylate functionalized with sodiumL-phenylalaninate is obtained.

According to the dry extract: [compound 7]=14.9 mg/g

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.65per saccharide unit.

The degree of substitution with sodium methylcarboxylates is 2.65 persaccharide unit.

AA8. Compound 8: Sodium Maltopentaosemethylcarboxylate Functionalizedwith Sodium L-Phenylalaninate

Via a process similar to that described for the preparation of compound1, but performed with maltopentaose (CarboSynth), 10 g ofmaltopentaosemethylcarboxylic acid with a degree of substitution withmethylcarboxylic acid of 1.75 per saccharide unit are obtained and thenlyophilized.

Via a process similar to that used for the preparation of compound 1, asodium maltopentaosemethylcarboxylate functionalized with sodiumL-phenylalaninate is obtained.

According to the dry extract: [compound 8]=7.1 mg/g

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.75per saccharide unit.

The degree of substitution with sodium methylcarboxylates is 1.0 persaccharide unit.

AA9. Compound 9: Sodium Maltooctaosemethylcarboxylate Functionalizedwith Sodium L-Phenylalaninate

Via a process similar to that described for the preparation of compound1, but performed with maltooctaose (CarboSynth), 10 g ofmaltooctaosemethylcarboxylic acid with a degree of substitution withmethylcarboxylic acid of 1.65 per saccharide unit are obtained and thenlyophilized.

Via a process similar to that used for the preparation of compound 1, asodium maltooctaosemethylcarboxylate functionalized with sodiumL-phenylalaninate is obtained.

According to the dry extract: [compound 9]=26.3 mg/g

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.65per saccharide unit.

The degree of substitution with sodium methylcarboxylates is 1.0 persaccharide unit.

AA10. Compound 10: Sodium Maltotriosemethylcarboxylate Functionalizedwith Sodium L-Tyrosinate

Via a process similar to that described for the preparation of compound1, but performed with methyl L-tyrosinate, hydrochloride salt (Bachem),a sodium maltotriosemethylcarboxylate, characterized by a degree ofsubstitution with sodium methylcarboxylate per saccharide unit of 1.64,is functionalized with sodium L-tyrosinate.

According to the dry extract: [compound 10]=9.1 mg/g

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-tyrosinate is 0.81 persaccharide unit. The degree of substitution with sodiummethylcarboxylates is 0.83 per saccharide unit.

AA11. Compound 11: Sodium Maltotriosemethylcarboxylate Functionalizedwith Sodium Alpha-Phenylglycinate

Via a process similar to that described for the preparation of compound1, 10 g of maltotriosemethylcarboxylic acid with a degree ofsubstitution with methylcarboxylic acid of 1.64 per saccharide unit areobtained and then lyophilized.

8 g of maltotriosemethylcarboxylic acid (50 mmol of methylcarboxylicacid functions) are dissolved in DMF and then cooled to 0° C. A mixtureof sodium alpha-phenylglycinate (Bachem, 5 g; 33 mmol) and triethylamine(33 mmol) is prepared in water. A solution of NMM (4.9 g; 49 mmol) andof EtOCOCl (5.3 g, 49 mmol) is then added to the solution ofmaltotriosemethylcarboxylic acid at 0° C. The solution of sodiumalpha-phenylglycinate and triethylamine is then added and the mixture isstirred at 30° C. An aqueous imidazole solution (340 g/L) is added after90 minutes. The medium is diluted with water and the solution obtainedis then purified by ultrafiltration on a 1 kDa PES membrane against a150 mM NaHCO₃/Na₂CO₃ pH 10.4 buffer, 0.9% NaCl and water. The compoundconcentration of the final solution is determined on the dry extract. Asample of solution is lyophilized and analyzed by ¹H NMR in D₂O todetermine the degree of substitution with methylcarboxylatesfunctionalized with sodium alpha-phenylglycinate.

According to the dry extract: [compound 11]=9.1 mg/g

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium alpha-phenylglycinate is0.52 per saccharide unit.

The degree of substitution with sodium methylcarboxylates is 1.12 persaccharide unit.

AB Polysaccharide Counterexamples AB1. Polysaccharide 1: SodiumDextranmethylcarboxylate Functionalized with Sodium L-Phenylalaninate

Polysaccharide 1 is a sodium dextranmethylcarboxylate functionalizedwith sodium L-phenylalaninate obtained from a dextran with aweight-average molar mass of 10 kg/mol (DP=39, Pharmacosmos) accordingto the process described in patent application FR 07/02316 publishedunder the number FR 2 914 305. According to the acid/base assay, thedegree of substitution with sodium methylcarboxylates is 0.6 persaccharide unit.

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.46per saccharide unit.

This polysaccharide corresponds to polysaccharide 1 of patentapplication FR 09/01478.

AB2. Polysaccharide 2: Sodium Dextranmethylcarboxylate Functionalizedwith Sodium L-Phenylalaninate

Polysaccharide 2 is a sodium dextranmethylcarboxylate functionalizedwith sodium L-phenylalaninate obtained from a dextran with aweight-average molar mass of 5 kg/mol (DP=19, Pharmacosmos) according tothe process described in patent application FR 07/02316 published underthe number FR 2 914 305. According to the acid/base assay, the degree ofsubstitution with sodium methylcarboxylates is 1.01 per saccharide unit.

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.64per saccharide unit.

AB3. Polysaccharide 3: Sodium Dextranmethylcarboxylate Functionalizedwith Sodium L-Phenylalaninate

Polysaccharide 3 is a sodium dextranmethylcarboxylate functionalizedwith sodium L-phenylalaninate obtained from a dextran with aweight-average molar mass of 5 kg/mol (DP=19, Pharmacosmos) according tothe process described in patent application FR 07/02316 published underthe number FR 2 914 305. According to the acid/base assay, the degree ofsubstitution with sodium methylcarboxylates is 0.65 per saccharide unit.

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.45per saccharide unit.

AB4. Polysaccharide 4: Sodium Dextranmethylcarboxylate Functionalizedwith Sodium L-Phenylalaninate

Polysaccharide 4 is a sodium dextranmethylcarboxylate functionalizedwith sodium L-phenylalaninate obtained from a dextran with aweight-average molar mass of 10 kg/mol (DP=39, Pharmacosmos) accordingto the process described in patent application FR 07/02316 publishedunder the number FR 2 914 305. According to the acid/base assay, thedegree of substitution with sodium methylcarboxylates is 1.01 persaccharide unit.

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.64per saccharide unit.

AB5. Polysaccharide 5: Sodium Dextranmethylcarboxylate Functionalizedwith Sodium L-Phenylalaninate

Polysaccharide 5 is a sodium dextranmethylcarboxylate functionalizedwith sodium L-phenylalaninate obtained from a dextran with aweight-average molar mass of 5 kg/mol (DP=19, Pharmacosmos) according tothe process described in patent application FR 07/02316 published underthe number FR 2 914 305. According to the acid/base assay, the degree ofsubstitution with sodium methylcarboxylates is 0.45 per saccharide unit.

According to the ¹H NMR: the degree of substitution withmethylcarboxylates functionalized with sodium L-phenylalaninate is 0.65per saccharide unit.

AC Polyanionic Compound Polyanionic Compound 1: SodiumMaltotriosemethylcarboxylate

To 8 g (143 mmol of hydroxyl functions) of maltotriose (CarboSynth)dissolved in water at 65° C. is added 0.6 g (16 mmol) of sodiumborohydride. After stirring for 30 minutes, 28 g (238 mmol) of sodiumchloroacetate are added. To this solution are then added dropwise 24 mLof 10 N NaOH (240 mmol), and the mixture is then heated at 65° C. for 90minutes. 16.6 g (143 mmol) of sodium chloroacetate are then added to thereaction medium, along with dropwise addition of 14 mL of 10 N NaOH (140mmol). After heating for 1 hour, the mixture is diluted with water,neutralized with acetic acid and then purified by ultrafiltration on a 1kDa PES membrane against water. The compound concentration of the finalsolution is determined on the dry extract, and an acid/base assay in a50/50 (V/V) water/acetone mixture is then performed to determine thedegree of substitution with sodium methylcarboxylate.

According to the dry extract: [polyanionic compound 1]=32.9 mg/g

According to the acid/base assay: the degree of substitution with sodiummethylcarboxylates is 1.65 per saccharide unit.

B Preparation of the Solutions B1. Solution of Rapid Insulin AnalogNovolog® at 100 IU/mL

This solution is a commercial solution of aspart insulin from NovoNordisk sold under the name Novolog®. This product is an aspart rapidinsulin analog.

B2. Solution of Rapid Insulin Analog Humalog® at 100 IU/mL

This solution is a commercial solution of lispro insulin from Eli Lillysold under the name Humalog®. This product is a rapid insulin analog.

B3. Solution of Regular Human Insulin Actrapid® at 100 IU/mL

This solution is a commercial solution of human insulin from NovoNordisk sold under the name Actrapid®. This product is a regular humaninsulin.

B4. Solution of Regular Human Insulin Humulin® R at 100 IU/mL

This solution is a commercial solution of human insulin from Eli Lillysold under the name Humulin® R. This product is a regular human insulin.

B5. Preparation of the Excipient Solutions

The non-polymeric polyanionic compounds are selected by measuring theirdissociation constant with respect to calcium ions and with respect totheir capacity for not destabilizing the hexameric form of insulin.

As regards the dissociation constant with respect to calcium ions, it isdetermined as follows.

Solutions containing 2.5 mM of CaCl₂, 150 mM of NaCl and increasingconcentrations of polyanionic compound (between 0 and 20 mM) areprepared. The potential of all these formulations is measured and theconcentrations of free calcium ions in the formulations are determined.After linearization by the Scatchard method, the dissociation constantsare established. These data make it possible to compare the affinity ofthe carboxylates and phosphates of the various polyanionic compounds forCa.

As regards their capacity for not destabilizing the hexameric form ofinsulin, this property is measured by circular dichroism in comparisonwith insulin alone (without anionic compound or polyanionic compound),see the experimental protocols in experimental section D.

Preparation of a Sodium Citrate Solution at 1.188 M

A sodium citrate solution is obtained by dissolving 9.0811 g of sodiumcitrate (30.9 mmol) in 25 mL of water in a graduated flask. The pH isadjusted to exactly 7.4 by adding 1 mL of 1 M HCl. The solution isfiltered through a 0.22 μm filter.

Preparation of a 130 mM m-Cresol Solution

An m-cresol solution is obtained by dissolving 14.114 g of m-cresol (130mmol) in 986.4 mL of water in a 1 L graduated flask.

Preparation of a Solution of m-Cresol and Glycerol (96.6 mM m-Cresol and566 mM Glycerol)

73.3 g of the 130 mM m-cresol solution are added to 5.26 g of glyceroland then diluted by addition of 22.25 g of water. The m-cresol andglycerol solution obtained is homogenized for 30 minutes and thenfiltered through a 0.22 μm membrane.

Preparation of a 32.7 mM Tween 20 Solution

A Tween 20 solution is obtained by dissolving 2.0079 g of Tween 20(1.636 mmol) in 50 mL of water in a graduated flask. The solution isfiltered through a 0.22 μm membrane.

B6. Preparation of a 500 IU/mL Human Insulin Solution

15 g of water are added to 563.6 mg of human insulin and the pH is thenlowered to acidic pH by adding 5.98 g of 0.1 N HCl. After totaldissolution of the insulin at acidic pH, the solution is neutralized topH 7.2 by adding 8.3 mL of 0.1 N NaOH. The concentration is thenadjusted to 500 IU/mL by adding 0.76 g of water. The solution is finallyfiltered through a 0.22 μm membrane.

B7. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 1

For a final volume of 100 mL of formulation, with a [compound 1]/[lisproinsulin] mass ratio of 2.0, the various reagents are added in theamounts specified below and in the following order:

Lyophilized compound 1 730 mg 100 IU/mL Humalog ® commercial solution100 mL

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B8. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[lisproinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 1 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B9. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound1]/[polyanionic compound 1]/[lispro insulin] mass ratio of 2.0/2.0/1,the various reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 1 730 mg Lyophilized polyanionic compound 1 730 mg100 IU/mL Humalog ® commercial solution 100 mL

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B10. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound1]/[polyanionic compound 1]/[lispro insulin] mass ratio of 2.0/5.5/1,the various reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 1 730 mg Lyophilized polyanionic compound 1 2000 mg100 IU/mL Humalog ® commercial solution 100 mL

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B11. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[lisproinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 2 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B12. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound2]/[polyanionic compound 1]/[lispro insulin] mass ratio of 2.0/2.0/1,the various reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 2 730 mg Lyophilized polyanionic compound 1 730 mg100 IU/mL Humalog ® commercial solution 100 mL

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B13. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound2]/[polyanionic compound 1]/[lispro insulin] mass ratio of 2.0/5.5/1,the various reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 2 730 mg Lyophilized polyanionic compound 1 2000 mg100 IU/mL Humalog ® commercial solution 100 mL

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B14. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 1

For a final volume of 100 mL of formulation, with a [compound 1]/[lisproinsulin] mass ratio of 4, the various reagents are added in the amountsspecified below:

Compound 1 in lyophilized form 1460 mg 100 IU/mL Humalog ® commercialsolution 100 mL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B15. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 2

For a final volume of 100 mL of formulation, with a [compound 2]/[lisproinsulin] mass ratio of 4, the various reagents are added in the amountsspecified below:

Compound 2 in lyophilized form 1460 mg 100 IU/mL Humalog ® commercialsolution 100 mL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B16. Preparation of a 100 IU/mL Lispro Insulin Analog Solution in thePresence of Compound 1 and Sodium Tartrate

For a final volume of 100 mL of formulation, with a [compound 1]/[lisproinsulin] mass ratio of 2.0 and a sodium tartrate concentration of 80 mM,the various reagents are added in the amounts specified below:

Compound 1 in lyophilized form 730 mg 100 IU/mL Humalog ® commercialsolution 100 mL Sodium tartrate 1.552 g

For the tartrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B17. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound1]/[polyanionic compound 1]/[lispro insulin] mass ratio of 2/4/1, thevarious reagents are added in the amounts specified below:

Compound 1 in lyophilized form 730 mg Polyanionic compound 1 inlyophilized form 1460 mg 100 IU/mL Humalog ® commercial solution 100 mL

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B18. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and Sodium Triphosphate

For a final volume of 100 mL of formulation, the various reagents areadded in the amounts specified below:

Compound 1 in lyophilized form 730 mg Sodium triphosphate 184 mg 100IU/mL Humalog ® commercial solution 100 mL

For the triphosphate, use may be made of the acid form or the basic formin the form of the sodium salt, the potassium salt or another salt thatis compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B19. Preparation of a 100 IU/mL Lispro Insulin Analog Solution in thePresence of Compound 2 and Sodium Tartrate

For a final volume of 100 mL of formulation, with a [compound 2]/[lisproinsulin] mass ratio of 2.0 and a sodium tartrate concentration of 80 mM,the various reagents are added in the amounts specified below:

Compound 2 in lyophilized form 730 mg 100 IU/mL Humalog ® commercialsolution 100 mL Sodium tartrate 1.552 g

For the tartrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B20. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound2]/[polyanionic compound 1]/[lispro insulin] mass ratio of 2/4/1, thevarious reagents are added in the amounts specified below:

Compound 2 in lyophilized form 730 mg Polyanionic compound 1 inlyophilized form 1460 mg 100 IU/mL Humalog ® commercial solution 100 mL

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B21. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and Sodium Triphosphate

For a final volume of 100 mL of formulation, the various reagents areadded in the amounts specified below:

Compound 2 in lyophilized form 730 mg Sodium triphosphate 184 mg 100IU/mL Humalog ® commercial solution 100 mL

For the triphosphate, use may be made of the acid form or the basic formin the form of the sodium salt, the potassium salt or another salt thatis compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B22. Preparation of a 200 IU/mL Insulin Analog (Lispro Insulin) Solution

The commercial formulation of lispro insulin (Humalog®) was concentratedusing Amicon Ultra-15 centrifugation tubes with a 3 kDa cut-offthreshold. The Amicon tubes were first rinsed with 12 mL of deionizedwater. 12 mL of the commercial formulation were centrifuged for 35minutes at 4000 g at 20° C. The volume of the retentate was measured andthe concentration thus estimated. All the retentates were pooled and theoverall concentration was estimated (>200 IU/mL).

The concentration of this concentrated lispro insulin solution wasadjusted to 200 IU/mL by adding the commercial lispro insulinformulation (Humalog®). The concentrated lispro insulin formulation hasthe same concentrations of excipients (m-cresol, glycerol, phosphate) asthe commercial formulation at 100 IU/mL.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B23. Preparation of a 200 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[lisproinsulin] mass ratio of 2, the various reagents are mixed in the amountsspecified below:

200 IU/mL lispro insulin 100 mL Lyophilizate of compound 1 1460 mg1.188M sodium citrate solution 1566 μL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B24. Preparation of a 200 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound1]/[polyanionic compound 1]/[lispro insulin] mass ratio of 2/2/1, thevarious reagents are mixed in the amounts specified below:

200 IU/mL lispro insulin 100 mL Lyophilizate of compound 1 1460 mgLyophilizate of polyanionic compound 1 1460 mg

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B25. Preparation of a 200 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound1]/[polyanionic compound 1]/[lispro insulin] mass ratio of 2/4/1, thevarious reagents are mixed in the amounts specified below:

200 IU/mL lispro insulin 100 mL Lyophilizate of compound 1 1460 mgLyophilizate of polyanionic compound 1 2920 mg

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B26. Preparation of a 200 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound2]/[polyanionic compound 1]/[lispro insulin] mass ratio of 2/4/1, thevarious reagents are mixed in the amounts specified below:

200 IU/mL lispro insulin 100 mL Lyophilizate of compound 2 1460 mgLyophilizate of polyanionic compound 1 2920 mg

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B27. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 1 and Tartrate

For a final volume of 100 mL of formulation, with a [compound 1]/[humaninsulin] mass ratio of 2 and 80 mM of tartrate, the various reagents aremixed in the amounts specified below:

500 IU/mL human insulin 20 mL 36.01 mg/mL solution of compound 1 20.27mL 96.6 mM m-cresol/566 mM glycerol solution 30 mL Water 28.95 mL Sodiumtartrate 1.552 g

For the tartrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is 7.4±0.4. This clear solution is filtered through a 0.22μm membrane and then placed at +4° C.

B28. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 1 and Triphosphate

For a final volume of 100 mL of formulation, the various reagents aremixed in the amounts specified below:

500 IU/mL human insulin 20 mL 36.01 mg/mL solution of compound 1 20.27mL 96.6 mM m-cresol/566 mM glycerol solution 30 mL Water 28.95 mL Sodiumtriphosphate 184 mg

For the triphosphate, use may be made of the acid form or the basic formin the form of the sodium salt, the potassium salt or another salt thatis compatible with an injectable formulation.

The final pH is 7.4±0.4. This clear solution is filtered through a 0.22μm membrane and then placed at +4° C.

B29. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 2 and Tartrate

For a final volume of 100 mL of formulation, with a [compound 2]/[humaninsulin] mass ratio of 2 and 80 mM of tartrate, the various reagents aremixed in the amounts specified below:

500 IU/mL human insulin 20 mL 36.01 mg/mL solution of compound 2 20.27mL 96.6 mM m-cresol/566 mM glycerol solution 30 mL Water 28.95 mL Sodiumtartrate 1.552 g

For the tartrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is 7.4±0.4.

This clear solution is filtered through a 0.22 μm membrane and thenplaced at +4° C.

B30. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 2 and Triphosphate

For a final volume of 100 mL of formulation, the various reagents aremixed in the amounts specified below:

500 IU/mL human insulin 20 mL 36.01 mg/mL solution of compound 2 20.27mL 96.6 mM m-cresol/566 mM glycerol solution 30 mL Water 28.95 mL Sodiumtriphosphate 184 mg

For the triphosphate, use may be made of the acid form or the basic formin the form of the sodium salt, the potassium salt or another salt thatis compatible with an injectable formulation.

The final pH is 7.4±0.4. This clear solution is filtered through a 0.22μm membrane and then placed at +4° C.

B31. Preparation of a 200 IU/mL Human Insulin Solution

The commercial formulation of human insulin (Humulin® R) wasconcentrated using Amicon Ultra-15 centrifugation tubes with a 3 kDacut-off threshold. The Amicon tubes were first rinsed with 12 mL ofdeionized water. 12 mL of the commercial formulation were centrifugedfor 35 minutes at 4000 g at 20° C. The volume of the retentate wasmeasured and the concentration thus estimated. All the retentates werepooled and the overall concentration was estimated (>200 IU/mL).

The concentration of this concentrated human insulin solution wasadjusted to 200 IU/mL by adding the commercial human insulin formulation(Humulin® R). The concentrated human insulin formulation has the sameconcentrations of excipients (m-cresol, glycerol) as the commercialformulation at 100 IU/mL.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B32. Preparation of a 200 IU/mL Human Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[humaninsulin] mass ratio of 2, the various reagents are mixed in the amountsspecified below:

200 IU/mL human insulin 100 mL Lyophilizate of compound 1 1460 mg 1.188Msodium citrate solution 1566 μL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B33. Preparation of a 200 IU/mL Human Insulin Solution in the Presenceof Compound 1 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound1]/[polyanionic compound 1]/[human insulin] mass ratio of 2/2/1, thevarious reagents are mixed in the amounts specified below:

200 IU/mL human insulin 100 mL Lyophilizate of compound 1 1460 mgLyophilizate of polyanionic compound 1 1460 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B34. Preparation of a 200 IU/mL Human Insulin Solution in the Presenceof Compound 1 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound1]/[polyanionic compound 1]/[human insulin] mass ratio of 2/4/1, thevarious reagents are mixed in the amounts specified below:

200 IU/mL human insulin 100 mL Lyophilizate of compound 1 1460 mgLyophilizate of polyanionic compound 1 2920 mg

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B35. Preparation of a 200 IU/mL Human Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[humaninsulin] mass ratio of 2, the various reagents are mixed in the amountsspecified below:

200 IU/mL human insulin 100 mL Lyophilizate of compound 2 1460 mg 1.188Msodium citrate solution 1566 μL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B36. Preparation of a 200 IU/mL Human Insulin Solution in the Presenceof Compound 2 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound2]/[polyanionic compound 1]/[human insulin] mass ratio of 2/2/1, thevarious reagents are mixed in the amounts specified below:

200 IU/mL human insulin 100 mL Lyophilizate of compound 2 1460 mgLyophilizate of polyanionic compound 1 1460 mg

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B37. Preparation of a 200 IU/mL Human Insulin Solution in the Presenceof Compound 2 and the Polyanionic Compound 1

For a final volume of 100 mL of formulation, with a [compound2]/[polyanionic compound 1]/[human insulin] mass ratio of 2/4/1, thevarious reagents are mixed in the amounts specified below:

200 IU/mL human insulin 100 mL Lyophilizate of compound 2 1460 mgLyophilizate of polyanionic compound 1 2920 mg

The polyanionic compound 1 may be used in the acid form or the basicform in the form of the sodium salt, the potassium salt or another saltthat is compatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B38. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[humaninsulin] mass ratio of 2 and 9.3 mM of citrate, the various reagents aremixed in the amounts specified below:

500 IU/mL human insulin 20 mL 36.01 mg/mL solution of compound 2 20.27mL 96.6 mM m-cresol/566 mM glycerol solution 30 mL Water 28.95 mL 1.188Msodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is 7.4±0.4. This clear solution is filtered through a 0.22μm membrane and then placed at +4° C.

B39. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[humaninsulin] mass ratio of 2 and 9.3 mM of citrate, the various reagents aremixed in the amounts specified below:

500 IU/mL human insulin 20 mL 36.01 mg/mL solution of compound 1 27 mL96.6 mM m-cresol/566 mM glycerol solution 30 mL Water 28.95 mL 1.188Msodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is 7.4±0.4. This clear solution is filtered through a 0.22μm membrane and then placed at +4° C.

B40. Preparation of a 100 IU/mL Aspart Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[aspartinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 1 730 mg 100 IU/mL Novolog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B41. 100 IU/mL Solution of Rapid Insulin Analog Apidra®

This solution is a commercial solution of glulisine insulin fromSanofi-Aventis sold under the name Apidra®. This product is a rapidinsulin analog.

B42. Preparation of a 100 IU/mL Glulisine Insulin Solution in thePresence of Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound1]/[glulisine insulin] mass ratio of 2.0 and a citrate concentration of9.3 mM, the various reagents are added in the amounts specified belowand in the following order:

Lyophilized compound 1 730 mg 100 IU/mL Apidra ® commercial solution 100mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B43. Preparation of a 100 IU/mL Aspart Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[aspartinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 2 730 mg 100 IU/mL Novolog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B44. Preparation of a 100 IU/mL Glulisine Insulin Solution in thePresence of Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound2]/[glulisine insulin] mass ratio of 2.0 and a citrate concentration of9.3 mM, the various reagents are added in the amounts specified belowand in the following order:

Lyophilized compound 2 730 mg 100 IU/mL Apidra ® commercial solution 100mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B45. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 5 and Citrate

For a final volume of 100 mL of formulation, with a [compound 5]/[lisproinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 5 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B46. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 6 and Citrate

For a final volume of 100 mL of formulation, with a [compound 6]/[lisproinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 6 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B47. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 7 and Citrate

For a final volume of 100 mL of formulation, with a [compound 7]/[lisproinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 7 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B48. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 8 and Citrate

For a final volume of 100 mL of formulation, with a [compound 8]/[lisproinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 8 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B49. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 9 and Citrate

For a final volume of 100 mL of formulation, with a [compound 9]/[lisproinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 9 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

B50. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 5 and Citrate

For a final volume of 100 mL of formulation, with a [compound 5]/[humaninsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 5 730 mg 100 IU/mL Humulin ® R commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B51. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 6 and Citrate

For a final volume of 100 mL of formulation, with a [compound 6]/[humaninsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 6 730 mg 100 IU/mL Humulin ® R commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B52. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 7 and Citrate

For a final volume of 100 mL of formulation, with a [compound 7]/[humaninsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 7 730 mg 100 IU/mL Humulin ® R commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B53. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 8 and Citrate

For a final volume of 100 mL of formulation, with a [compound 8]/[humaninsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 8 730 mg 100 IU/mL Humulin ® R commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B54. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 9 and Citrate

For a final volume of 100 mL of formulation, with a [compound 9]/[humaninsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 9 730 mg 100 IU/mL Humulin ® R commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B55. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 2

For a final volume of 100 mL of formulation, with a [compound 2]/[humaninsulin] mass ratio of 2.0, the various reagents are added in theamounts specified below and in the following order:

Lyophilized compound 2 730 mg 100 IU/mL Humulin ® R commercial solution100 mL

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B56. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 7

For a final volume of 100 mL of formulation, with a [compound 7]/[humaninsulin] mass ratio of 2.0, the various reagents are added in theamounts specified below and in the following order:

Lyophilized compound 7 730 mg 100 IU/mL Humulin ® R commercial solution100 mL

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B57. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 10 and Citrate

For a final volume of 100 mL of formulation, with a [compound10]/[lispro insulin] mass ratio of 2.0 and a citrate concentration of9.3 mM, the various reagents are added in the amounts specified belowand in the following order:

Lyophilized compound 10 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B58. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 11 and Citrate

For a final volume of 100 mL of formulation, with a [compound11]/[lispro insulin] mass ratio of 2.0 and a citrate concentration of9.3 mM, the various reagents are added in the amounts specified belowand in the following order:

Lyophilized compound 11 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

B59. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 10 and Citrate

For a final volume of 100 mL of formulation, with a [compound 10]/[humaninsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 10 730 mg 100 IU/mL Humulin ® R commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B60. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 11 and Citrate

For a final volume of 100 mL of formulation, with a [compound 11]/[humaninsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 11 730 mg 100 IU/mL Humulin ® R commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B61. Preparation of a 200 IU/mL Aspart Insulin Solution

The commercial formulation of aspart insulin (Novolog®) was concentratedusing Amicon Ultra-15 centrifugation tubes with a 3 kDa cut-offthreshold. The Amicon tubes were first rinsed with 12 mL of deionizedwater. 12 mL of the commercial formulation were centrifuged for 35minutes at 4000 g at 20° C. The volume of the retentate was measured andthe concentration thus estimated. All the retentates were pooled and theoverall concentration was estimated (>200 IU/mL).

The concentration of this concentrated aspart insulin solution wasadjusted to 200 IU/mL by adding the commercial aspart insulinformulation (Novolog®). The concentrated aspart insulin formulation hasthe same concentrations of excipients (m-cresol, glycerol) as thecommercial formulation at 100 IU/mL.

By modifying the centrifugation time and the final dilution with thecommercial formulation, it is possible to prepare in the same manneraspart insulin formulations at 300, 400 or 500 IU/mL.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B62. Preparation of a 200 IU/mL Glulisine Insulin Solution

The commercial formulation of glulisine insulin (Apidra®) wasconcentrated using Amicon Ultra-15 centrifugation tubes with a 3 kDacut-off threshold. The Amicon tubes were first rinsed with 12 mL ofdeionized water. 12 mL of the commercial formulation were centrifugedfor 35 minutes at 4000 g at 20° C. The volume of the retentate wasmeasured and the concentration thus estimated. All the retentates werepooled and the overall concentration was estimated (>200 IU/mL).

The concentration of this concentrated glulisine insulin solution wasadjusted to 200 IU/mL by adding the commercial glulisine insulinformulation (Apidra®). The concentrated glulisine insulin formulationhas the same concentrations of excipients (m-cresol, NaCl, TRIS) as thecommercial formulation at 100 IU/mL.

By modifying the centrifugation time and the final dilution with thecommercial formulation, it is possible to prepare in the same mannerglulisine insulin formulations at 300, 400 or 500 IU/mL.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B63. Preparation of a 200 IU/mL Aspart Insulin Solution in the Presenceof Compound 1 at 14.6 mg/mL and 18.6 mM Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[aspartinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below and in the following order:

Lyophilizate of compound 1 1460 mg 200 IU/mL aspart insulin 100 mL1.188M sodium citrate solution 1566 μL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B64. Preparation of a Human Insulin, Lispro Insulin, Aspart Insulin orGlulisine Insulin Solution at 300, 400 and 500 IU/mL

Concentrated formulations of human insulin, lispro insulin, aspartinsulin or glulisine insulin at 300 IU/mL, 400 IU/mL or 500 IU/mL (andalso at all intermediate concentrations) are prepared on the basis ofthe protocol of example B62 relating to the preparation of a 200 IU/mLglulisine insulin solution. The commercial insulin formulation isconcentrated using Amicon Ultra-15 centrifugation tubes with a 3 kDacut-off threshold. The Amicon tubes are first rinsed with 12 mL ofdeionized water. 12 mL of the commercial formulation are centrifuged at4000 g at 20° C. By modifying the centrifugation time, it is possible toadjust the final concentration of insulin in the formulation. The volumeof the retentate is measured and the concentration is thus estimated.All the retentates are pooled and the overall concentration is estimated(>300, 400 or 500 IU/mL).

The concentration of this concentrated insulin solution is adjusted tothe desired concentration (e.g. 300 IU/mL, 400 IU/mL or 500 IU/mL) byaddition of the insulin formulation (Humulin® R, Novolog®, Humalog® orApidra®). The concentrated insulin formulation has the sameconcentrations of excipients as the commercial formulation at 100 IU/mL.

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B65. Preparation of a 200 IU/mL Glulisine Insulin Solution in thePresence of Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound1]/[glulisine insulin] mass ratio of 2, the various reagents are mixedin the amounts specified below and in the following order:

Lyophilizate of compound 1 1460 mg 200 IU/mL glulisine insulin 100 mL1.188M sodium citrate solution 1566 μL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B66. Preparation of a 300 IU/mL Aspart Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[aspartinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

300 IU/mL aspart insulin 100 mL Lyophilizate of compound 1 2190 mgSodium citrate 720 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B67. Preparation of a 300 IU/mL Glulisine Insulin Solution in thePresence of Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound1]/[glulisine insulin] mass ratio of 2.0, the various reagents are mixedin the amounts specified below:

300 IU/mL glulisine insulin 100 mL Lyophilizate of compound 1 2190 mgSodium citrate 720 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B68. Preparation of a 400 IU/mL Aspart Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[aspartinsulin] mass ratio of 2, the various reagents are mixed in the amountsspecified below:

400 IU/mL aspart insulin 100 mL Lyophilizate of compound 1 2920 mgSodium citrate 960 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B69. Preparation of a 400 IU/mL Glulisine Insulin Solution in thePresence of Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound1]/[glulisine insulin] mass ratio of 2.0, the various reagents are mixedin the amounts specified below:

400 IU/mL glulisine insulin 100 mL Lyophilizate of compound 1 2920 mgSodium citrate 960 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B70. Preparation of a 500 IU/mL Aspart Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[aspartinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

500 IU/mL aspart insulin 100 mL Lyophilizate of compound 1 3650 mgSodium citrate 1200 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B71. Preparation of a 500 IU/mL Glulisine Insulin Solution in thePresence of Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound1]/[glulisine insulin] mass ratio of 2.0, the various reagents are mixedin the amounts specified below:

500 IU/mL glulisine insulin 100 mL Lyophilizate of compound 1 3650 mgSodium citrate 1200 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B72. Preparation of a 500 IU/mL Glulisine insulin Solution in thePresence of Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[humaninsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

300 IU/mL human insulin 100 mL Lyophilizate of compound 1 2190 mg Sodiumcitrate 720 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B73. Preparation of a 300 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[lisproinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

300 IU/mL lispro insulin 100 mL Lyophilizate of compound 1 2190 mgSodium citrate 720 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B74. Preparation of a 400 IU/mL Human Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[humaninsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

400 IU/mL human insulin 100 mL Lyophilizate of compound 1 2920 mg Sodiumcitrate 960 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B75. Preparation of a 400 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[lisproinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

400 IU/mL lispro insulin 100 mL Lyophilizate of compound 1 2920 mgSodium citrate 960 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B76. Preparation of a 500 IU/mL Human Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[humaninsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

500 IU/mL human insulin 100 mL Lyophilizate of compound 1 3650 mg Sodiumcitrate 1200 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B77. Preparation of a 500 IU/mL Lispro Insulin Solution in the Presenceof Compound 1 and Citrate

For a final volume of 100 mL of formulation, with a [compound 1]/[lisproinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

500 IU/mL lispro insulin 100 mL Lyophilizate of compound 1 3650 mgSodium citrate 1200 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B78. Preparation of a 200 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[lisproinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

200 IU/mL lispro insulin 100 mL Lyophilizate of compound 2 1460 mg1.188M sodium citrate solution 1566 μL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B79. Preparation of a 200 IU/mL Aspart Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[aspartinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

200 IU/mL aspart insulin 100 mL Lyophilizate of compound 2 1460 mg1.188M sodium citrate solution 1566 μL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B80. Preparation of a 200 IU/mL Glulisine Insulin Solution in thePresence of Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound2]/[glulisine insulin] mass ratio of 2.0, the various reagents are mixedin the amounts specified below:

200 IU/mL glulisine insulin 100 mL Lyophilizate of compound 2 1460 mg1.188M sodium citrate solution 1566 μL

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B81. Preparation of a 300 IU/mL Aspart Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[aspartinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

300 IU/mL aspart insulin 100 mL Lyophilizate of compound 2 2190 mgSodium citrate 720 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B82. Preparation of a 300 IU/mL Glulisine Insulin Solution in thePresence of Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound2]/[glulisine insulin] mass ratio of 2.0, the various reagents are mixedin the amounts specified below:

300 IU/mL glulisine insulin 100 mL Lyophilizate of compound 2 2190 mgSodium citrate 720 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B83. Preparation of a 400 IU/mL Aspart Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[aspartinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

400 IU/mL aspart insulin 100 mL Lyophilizate of compound 2 2920 mgSodium citrate 960 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B84. Preparation of a 400 IU/mL Glulisine Insulin Solution in thePresence of Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound2]/[glulisine insulin] mass ratio of 2.0, the various reagents are mixedin the amounts specified below:

400 IU/mL glulisine insulin 100 mL Lyophilizate of compound 2 2920 mgSodium citrate 960 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B85. Preparation of a 500 IU/mL Aspart Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[aspartinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

500 IU/mL aspart insulin 100 mL Lyophilizate of compound 2 3650 mgSodium citrate 1200 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B86. Preparation of a 500 IU/mL Glulisine Insulin Solution in thePresence of Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound2]/[glulisine insulin] mass ratio of 2.0, the various reagents are mixedin the amounts specified below:

500 IU/mL glulisine insulin 100 mL Lyophilizate of compound 2 3650 mgSodium citrate 1200 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B87. Preparation of a 500 IU/mL Glulisine Insulin Solution in thePresence of Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[humaninsulin] mass ratio of 2, the various reagents are mixed in the amountsspecified below:

300 IU/mL human insulin 100 mL Lyophilizate of compound 2 2190 mg Sodiumcitrate 720 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B88. Preparation of a 300 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[lisproinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

300 IU/mL lispro insulin 100 mL Lyophilizate of compound 2 2190 mgSodium citrate 720 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B89. Preparation of a 400 IU/mL Human Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[humaninsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

400 IU/mL human insulin 100 mL Lyophilizate of compound 2 2920 mg Sodiumcitrate 960 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B90. Preparation of a 400 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[lisproinsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

400 IU/mL lispro insulin 100 mL Lyophilizate of compound 2 2920 mgSodium citrate 960 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B91. Preparation of a 500 IU/mL Human Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[humaninsulin] mass ratio of 2.0, the various reagents are mixed in theamounts specified below:

500 IU/mL human insulin 100 mL Lyophilizate of compound 2 3650 mg Sodiumcitrate 1200 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B92. Preparation of a 500 IU/mL Lispro Insulin Solution in the Presenceof Compound 2 and Citrate

For a final volume of 100 mL of formulation, with a [compound 2]/[lisproinsulin] mass ratio of 2.0, the various reagents are added in theamounts specified below:

500 IU/mL lispro insulin 100 mL Lyophilizate of compound 2 3650 mgSodium citrate 1200 mg

The final pH is adjusted to 7.4±0.4. The clear solution is filteredthrough a 0.22 μm membrane and stored at 4° C.

B93. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 3 and Citrate

For a final volume of 100 mL of formulation, with a [compound 3]/[lisproinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 3 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B94. Preparation of a 100 IU/mL Lispro Insulin Solution in the Presenceof Compound 4 and Citrate

For a final volume of 100 mL of formulation, with a [compound 4]/[lisproinsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 4 730 mg 100 IU/mL Humalog ® commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B95. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 3 and Citrate

For a final volume of 100 mL of formulation, with a [compound 3]/[humaninsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 3 730 mg 100 IU/mL Humulin ® R commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

B96. Preparation of a 100 IU/mL Human Insulin Solution in the Presenceof Compound 4 and Citrate

For a final volume of 100 mL of formulation, with a [compound 4]/[humaninsulin] mass ratio of 2.0 and a citrate concentration of 9.3 mM, thevarious reagents are added in the amounts specified below and in thefollowing order:

Lyophilized compound 4 730 mg 100 IU/mL Humulin ® R commercial solution100 mL 1.188M sodium citrate solution 783 μL

For the citrate, use may be made of the acid form or the basic form inthe form of the sodium salt, the potassium salt or another salt that iscompatible with an injectable formulation.

The final pH is adjusted to 7.4±0.4.

The clear solution is filtered through a 0.22 μm membrane and stored at4° C.

C Pharmacodynamics and Pharmacokinetics C1: Protocol for Measuring thePharmacodynamics of the Insulin Solutions

Twelve domestic pigs weighing about 50 kg, catheterized beforehand inthe jugular vein, are fasted for 2.5 hours before the start of theexperiment. In the hour preceding the injection of insulin, three bloodsamples are taken in order to determine the basal level of glucose andof insulin.

The injection of insulin at a dose of 0.09 IU/kg for lispro insulin andat a dose of 0.125 IU/kg for human insulin and aspart insulin isperformed subcutaneously into the neck, under the animal's ear, using aNovopen insulin pen equipped with a 31 G needle.

Blood samples are then taken every 4 minutes for 20 minutes and thenevery 10 minutes up to 3 hours. After taking each sample, the catheteris rinsed with a dilute heparin solution.

A drop of blood is taken to determine the glycemia using a glucometer.

The glucose pharmacodynamic curves are then plotted and the timerequired to reach the minimum glucose level in the blood for each pig isdetermined and reported as the glucose Tmin. The mean of the glucoseTmin values is then calculated.

The remaining blood is collected in a dry tube and is centrifuged toisolate the serum. The insulin levels in the serum samples are measuredvia the sandwich ELISA immunoenzymatic method for each pig.

The pharmacokinetic curves are then plotted. The time required to reachthe maximum insulin concentration in the serum for each pig isdetermined and reported as the insulin Tmax. The mean of the insulinTmax values is then calculated.

C2: Pharmacodynamic and Pharmacokinetic Results for the InsulinSolutions of Examples B2 and B8

Polyanionic Number of Example Insulin Compound compound pigs B2 Lispro —— 11 B8 Lispro 1 Citrate 9.3 mM 10

The pharmacodynamic results obtained with the formulations described inexamples B2 and B8 are presented in FIG. 1. According to the invention,the analysis of these curves shows that the formulation of example B8comprising compound 1 and citrate at 9.3 mM as excipient (curve plottedwith the squares corresponding to example B8, glucose Tmin=30±11 min)makes it possible to obtain more rapid action than that obtained withthe Humalog® commercial formulation of example B2 (curve plotted withthe triangles corresponding to example B2, glucose Tmin=44±14 min).

The pharmacokinetic results obtained with the formulations described inexamples B2 and B8 are presented in FIG. 2. According to the invention,the analysis of these curves shows that the formulation of example B8comprising compound 1 and citrate at 9.3 mM as excipients (curve plottedwith the squares corresponding to example B8, insulin Tmax=11±6 min)induces more rapid absorption of the lispro insulin than that of theHumalog® commercial formulation of example B2 (curve plotted with thetriangles corresponding to example B2, insulin Tmax=18±8 min).

C3: Pharmacodynamic and Pharmacokinetic Results for the InsulinSolutions of Examples B2 and B10

Polyanionic Number of Example Insulin Compound compound pigs B2 Lispro —— 11 B10 Lispro 1 Polyanionic 11 compound 1

The pharmacodynamic results obtained with the formulations described inexamples B2 and B10 are presented in FIG. 3. According to the invention,the analysis of these curves shows that the formulation of example B10comprising compound 1 and the polyanionic compound 1 as excipients at 20mg/mL (curve plotted with the squares corresponding to example B10,glucose Tmin=33±13 min) makes it possible to obtain more rapid actionthan that obtained with the Humalog® commercial formulation of exampleB2 (curve plotted with the triangles corresponding to example B2,glucose Tmin=44±14 min).

The pharmacokinetic results obtained with the formulations described inexamples B2 and B10 are presented in FIG. 4. According to the invention,the analysis of these curves shows that the formulation of example B10comprising compound 1 and the polyanionic compound 1 as excipients at 20mg/mL (curve plotted with the squares corresponding to example B10,insulin Tmax=15±9 min) induces more rapid absorption of the lisproinsulin than that of the Humalog® commercial formulation of example B2(curve plotted with the triangles corresponding to example B2, insulinTmax=18±8 min).

C4: Pharmacodynamic and Pharmacokinetic Results for the InsulinSolutions of Examples B2 and B7

Polyanionic Number of Example Insulin Compound compound pigs B2 Lispro —— 12 B7 Lispro 1 — 12

The pharmacodynamic results obtained with the formulations described inexamples B2 and B7 are presented in FIG. 5. According to the invention,the analysis of these curves shows that the formulation of example B7comprising compound 1 as excipient (curve plotted with the squarescorresponding to example B7, glucose Tmin=41±16 min) includes more rapidonset of action than that obtained with the Humalog® commercialformulation of example B2 (curve plotted with the trianglescorresponding to example B2, glucose Tmin=50±14 min).

The pharmacokinetic results obtained with the formulations described inexamples B2 and B7 are presented in FIG. 6. The analysis of these curvesshows that the formulation comprising compound 1 as excipient (curveplotted with the squares corresponding to example B2, insulin Tmax=21±10min) does not induce more rapid absorption of the lispro insulin thanthat of the Humalog® commercial formulation of example B2 (curve plottedwith the triangles corresponding to example B2 (insulin Tmax=20±9 min).Compound 1 alone is therefore insufficient to induce significantacceleration of the pharmacokinetics of lispro insulin.

C5: Pharmacodynamic and Pharmacokinetic Results for the InsulinSolutions of Examples B1 and B3

Polyanionic Number of Example Insulin Compound compound pigs B1 Aspart —— 11 B3 Human — — 11

The pharmacodynamic results obtained with the formulations described inexamples B1 and B3 are presented in FIG. 7. The analysis of these curvesshows that the human insulin formulation of example B3 (curve plottedwith the squares corresponding to example B3, glucose Tmin=61±31 min)does indeed have slower action than that of the aspart insulincommercial formulation of example B1 (curve plotted with the trianglescorresponding to example B1, glucose Tmin=44±13 min).

The pharmacokinetic results obtained with the formulations described inexamples B1 and B3 are presented in FIG. 8. The analysis of these curvesshows that the human insulin formulation alone of example B3 (curveplotted with the squares corresponding to example B3, insulin Tmax=36±33min) does indeed induce slower absorption than that of the aspartinsulin commercial formulation (Novolog®) of example B1 (curve plottedwith the triangles corresponding to example B1, insulin Tmax=28±13 min).

These results are in accordance with the literature results, withacceleration of the lowering of glycemia and of the absorption ofinsulin for a rapid insulin analog relative to a human insulin.

C6: Pharmacodynamic and Pharmacokinetic Results for the InsulinSolutions of Examples B1 and B39

Polyanionic Number of Example Insulin Compound compound pigs B1 Aspart —— 14 B39 Human 1 Citrate 9.3 mM 5

The pharmacodynamic results obtained with the formulations described inexamples B1 and B39 are presented in FIG. 9. The analysis of thesecurves shows that the formulation based on human insulin of example B39comprising compound 1 and citrate at 9.3 mM as excipients (curve plottedwith the squares corresponding to example B39, glucose Tmin=46±9 min)makes it possible to obtain similar action to that obtained with theaspart insulin commercial formulation (Novolog®) of example B1 (curveplotted with the triangles corresponding to example B1, glucoseTmin=53±24 min).

The pharmacokinetic results obtained with the formulations described inexamples B1 and B39 are presented in FIG. 10. The analysis of thesecurves shows that the formulation of example B39 comprising compound 1and citrate at 9.3 mM as excipients (curve plotted with the squarescorresponding to example B39, insulin Tmax=20±7 min) induces insulinabsorption similar to that obtained with the aspart insulin commercialformulation (Novolog®) of example B1 (curve plotted with the trianglescorresponding to example B1, insulin Tmax=22±10 min).

Since the time parameters for the aspart insulin (Novolog®) betweenexamples C5 and C6 are similar, it may be deduced by extrapolation thatthe formulation of example B39 induces acceleration of the lowering ofglycemia and of the absorption of human insulin relative to thecommercial formulation of human insulin (example B3).

C7: Pharmacodynamic and Pharmacokinetic Results for the InsulinSolutions of Examples B2 and B11

Polyanionic Number of Example Insulin Compound compound pigs B2 Lispro —— 26 B11 Lispro 2 Citrate 9.3 mM 23

The pharmacodynamic results obtained with the formulations described inexamples B2 and B11 are presented in FIG. 13. According to theinvention, the analysis of these curves shows that the formulation ofexample B11 comprising compound 2 and citrate at 9.3 mM as excipients(curve plotted with the squares corresponding to example B11, glucoseTmin=32±10 min) makes it possible to obtain more rapid action than thatobtained with the Humalog commercial formulation of example B2 (curveplotted with the triangles corresponding to example B2, glucoseTmin=41±21 min).

The pharmacokinetic results obtained with the formulations described inexamples B2 and B11 are presented in FIG. 14. According to theinvention, the analysis of these curves shows that the formulation ofexample B11 comprising compound 2 and citrate at 9.3 mM as excipients(curve plotted with the squares corresponding to example B11, insulinTmax=13±5 min) induces more rapid absorption of the lispro insulin thanthat of the Humalog® commercial formulation of example B2 (curve plottedwith the triangles corresponding to example B2, insulin Tmax=22±13 min).

C8: Pharmacodynamic and Pharmacokinetic Results for the InsulinSolutions of Examples B1 and B38

Polyanionic Number of Example Insulin Compound compound pigs B1 Aspart —— 37 B38 Human 2 Citrate 9.3 mM 31

The pharmacodynamic results obtained with the formulations described inexamples B1 and B38 are presented in FIG. 15. The analysis of thesecurves shows that the formulation based on human insulin of example B38comprising compound 2 and citrate at 9.3 mM as excipients (curve plottedwith the squares corresponding to example B102, glucose Tmin=47±30 min)makes it possible to obtain similar action to that obtained with theaspart insulin commercial formulation (Novolog®) of example B1 (curveplotted with the triangles corresponding to example B1, glucoseTmin=47±15 min).

The pharmacokinetic results obtained with the formulations described inexamples B1 and B38 are presented in FIG. 16. The analysis of thesecurves shows that the formulation of example B38 comprising compound 2and citrate at 9.3 mM as excipients (curve plotted with the squarescorresponding to example B38, insulin Tmax=22±21 min) induces humaninsulin absorption similar to that obtained with the aspart insulincommercial formulation (Novolog®) of example B1 (curve plotted with thetriangles corresponding to example B1, insulin Tmax=19±12 min).

Since the time parameters for the aspart insulin (Novolog®) betweenexamples C5 and C8 are similar, it may be deduced by extrapolation thatthe formulation of example B38 induces acceleration of the lowering ofglycemia and of the absorption of human insulin relative to thecommercial formulation of human insulin (example B3).

C9: Pharmacodynamic and Pharmacokinetic Results for the InsulinSolutions of Examples B1 and B53

Polyanionic Number of Example Insulin Compound compound pigs B1 Aspart —— 12 B53 Human Compound 8 Citrate 9.3 mM 8

The pharmacodynamic results obtained with the formulations described inexamples B1 and B53 are presented in FIG. 17. The analysis of thesecurves shows that the formulation based on human insulin of example B53comprising compound 8 and citrate at 9.3 mM as excipients (curve plottedwith the squares corresponding to example B53, glucose Tmin=63±36 min)makes it possible to obtain action virtually as rapid as that obtainedwith the aspart insulin commercial formulation (Novolog®) of example B1(curve plotted with the triangles corresponding to example B1, glucoseTmin=53±19 min).

The pharmacokinetic results obtained with the formulations described inexamples B1 and B53 are presented in FIG. 18. The analysis of thesecurves shows that the formulation of example B53 comprising compound 8and citrate at 9.3 mM as excipients (curve plotted with the squarescorresponding to example B53, insulin Tmax=19±12 min) induces humaninsulin absorption similar to that obtained with the aspart insulincommercial formulation (Novolog®) of example B1 (curve plotted with thetriangles corresponding to example B1, insulin Tmax=19±6 min).

Since the time parameters for the aspart insulin (Novolog®) betweenexamples C5 and C9 are similar, it may be deduced by extrapolation thatthe formulation of example B53 induces acceleration of the lowering ofglycemia and of the absorption of human insulin relative to thecommercial formulation of human insulin (example B3).

D Circular Dichroism D1: Association State of Lispro Insulin Evaluatedby Circular Dichroism in the Presence of Compound 1

Circular dichroism makes it possible to study the secondary andquaternary structure of insulin. The insulin monomers are organized asdimers and as hexamers. The hexamer is the physically and chemicallymost stable form of insulin. Two hexameric forms exist, the R6 form andthe T6 form. Lispro insulin has a strong CD signal at 251 nmcharacteristic of the R6 hexameric form (most stable form). Loss of theCD signal at 251 nm is linked to destabilization of the hexamer (andthus the first sign of transformation of the hexamer into dimer).

EDTA and the EDTA/citrate mixture completely destructure the R6 form oflispro insulin (FIG. 11). EDTA thus has a pronounced effect on thehexamer.

In contrast, citrate alone, compound 1 alone, and also the mixturecompound 1/citrate and compound 1/polyanionic compound 1, have virtuallyno impact on the CD signal at 251 nm. These compounds therefore havevirtually no impact on the R6 structure of the hexamer and, all the lessso, on the hexameric structure.

D2: Association State of Human Insulin Evaluated by Circular Dichroismin the Presence of Compound 1

Circular dichroism makes it possible to study the secondary andquaternary structure of insulin. The insulin monomers are organized asdimers and as hexamers. The hexamer is the physically and chemicallymost stable form of insulin. The CD signal at 275 nm is characteristicof the hexameric form of insulin (hexamer signal at about −300°, signalfor the dimer between −200° and −250°, and signal for the monomer below−200°. Loss of the CD signal at 275 nm is therefore characteristic ofdestabilization of the hexamer into dimers or monomers.

EDTA and the EDTA/citrate combination have a very pronounced impact onthe hexameric structure of human insulin (total dissociation of thehexamer into dimers, FIG. 12). In contrast, citrate alone, compound 1alone, the polyanionic compound 1 alone and also the compound 1/citrateand compound 1/polyanionic compound 1 combinations have no impact on thehexameric structure of human insulin. Unlike EDTA, the human insulinformulations comprising compound 1 and citrate or the polyanioniccompound 1 do not show any dissociation of the human insulin hexamer.

D3: Association State of Lispro Insulin Evaluated by Circular Dichroismin the Presence of Compounds 1 to 11

Circular dichroism makes it possible to study the secondary andquaternary structure of insulin. The insulin monomers are organized asdimers and as hexamers. The hexamer is the physically and chemicallymost stable form of insulin. Two hexameric forms exist, the R6 form andthe T6 form. Lispro insulin has a strong CD signal at 251 nmcharacteristic of the R6 hexameric form (most stable form). Loss of theCD signal at 251 nm is linked to destabilization of the hexamer (andthus the first sign of transformation of the hexamer into dimer). Theresults obtained are presented in FIG. 19. This figure describes on thex-axis:

-   -   A: lispro insulin (100 IU/mL)    -   B: lispro insulin+7.3 mg/mL of compound 2    -   C: lispro insulin+7.3 mg/mL of compound 2+citrate at 9.3 mM    -   D: lispro insulin+7.3 mg/mL of compound 1    -   E: lispro insulin+7.3 mg/mL of compound 1+citrate at 9.3 mM    -   F: lispro insulin+7.3 mg/mL of compound 3    -   G: lispro insulin+7.3 mg/mL of compound 3+citrate at 9.3 mM    -   H: lispro insulin+7.3 mg/mL of compound 4    -   I: lispro insulin+7.3 mg/mL of compound 4+citrate at 9.3 mM    -   J: lispro insulin+7.3 mg/mL of compound 5    -   K: lispro insulin+7.3 mg/mL of compound 5+citrate at 9.3 mM    -   L: lispro insulin+7.3 mg/mL of compound 6    -   M: lispro insulin+7.3 mg/mL of compound 6+citrate at 9.3 mM    -   N: lispro insulin+7.3 mg/mL of compound 7    -   O: lispro insulin+7.3 mg/mL of compound 7+citrate at 9.3 mM    -   P: lispro insulin+7.3 mg/mL of compound 8    -   Q: lispro insulin+7.3 mg/mL of compound 8+citrate at 9.3 mM    -   R: lispro insulin+7.3 mg/mL of compound 9    -   S: lispro insulin+7.3 mg/mL of compound 9+citrate at 9.3 mM    -   T: lispro insulin+7.3 mg/mL of compound 10    -   U: lispro insulin+7.3 mg/mL of compound 10+citrate at 9.3 mM    -   V: lispro insulin+7.3 mg/mL of compound 11    -   W: lispro insulin+7.3 mg/mL of compound 11+citrate at 9.3 mM        and on the y-axis the circular dichroism signal at 251 nm        (deg·cm²·dmol⁻¹).

Compounds 1 to 11 alone and also compounds 1 to 11 in combination withcitrate have no impact on the CD signal at 251 nm for lispro insulin.Compounds 1 to 11 therefore have no impact on the R6 structure of thehexamer and, all the less so, on the hexameric structure of lisproinsulin.

D4: Association State of Human Insulin Evaluated by Circular Dichroismin the Presence of Compounds 1 to 11

Circular dichroism makes it possible to study the secondary andquaternary structure of insulin. The insulin monomers are organized asdimers and as hexamers. The hexamer is the physically and chemicallymost stable form of insulin. The CD signal at 275 nm is characteristicof the hexameric form of insulin (hexamer signal at about −300°, signalfor the dimer between −200° and −250°, and signal for the monomer below−200°). Loss of the CD signal at 275 nm is therefore characteristic ofdestabilization of the hexamer into dimers or monomers. The resultsobtained are presented in FIG. 20. This figure describes on the x-axis:

A: human insulin (100 IU/mL)

B: human insulin+7.3 mg/mL of compound 2

C: human insulin+7.3 mg/mL of compound 2+citrate at 9.3 mM

D: human insulin+7.3 mg/mL of compound 1

E: human insulin+7.3 mg/mL of compound 1+citrate at 9.3 mM

F: human insulin+7.3 mg/mL of compound 3

G: human insulin+7.3 mg/mL of compound 3+citrate at 9.3 mM

H: human insulin+7.3 mg/mL of compound 4

I: human insulin+7.3 mg/mL of compound 4+citrate at 9.3 mM

J: human insulin+7.3 mg/mL of compound 5

K: human insulin+7.3 mg/mL of compound 5+citrate at 9.3 mM

L: human insulin+7.3 mg/mL of compound 6

M: human insulin+7.3 mg/mL of compound 6+citrate at 9.3 mM

N: human insulin+7.3 mg/mL of compound 7

O: human insulin+7.3 mg/mL of compound 7+citrate at 9.3 mM

P: human insulin+7.3 mg/mL of compound 8

Q: human insulin+7.3 mg/mL of compound 8+citrate at 9.3 mM

R: human insulin+7.3 mg/mL of compound 9

S: human insulin+7.3 mg/mL of compound 9+citrate at 9.3 mM

T: human insulin+7.3 mg/mL of compound 10

U: human insulin+7.3 mg/mL of compound 10+citrate at 9.3 mM

V: human insulin+7.3 mg/mL of compound 11

W: human insulin+7.3 mg/mL of compound 11+citrate at 9.3 mM

and on the y-axis the circular dichroism signal at 275 nm(deg·cm²·dmol⁻¹).

Compounds 1 to 11 alone and also compounds 1 to 11 in combination withcitrate have no impact on the CD signal at 275 nm for human insulin.Compounds 1 to 11 therefore have no impact on the hexameric structure ofhuman insulin.

E Dissolution of Human Insulin and Insulin Analogs at the IsoelectricPoint E1. Dissolution of Human Insulin at its Isoelectric Point

Human insulin has an isoelectric point at 5.3. At this pH of 5.3, humaninsulin precipitates. A test demonstrating the formation of a complex ofhuman insulin with the various compounds is performed at the isoelectricpoint. If an interaction exists, it is possible to dissolve the insulinat its isoelectric point.

A 200 IU/mL human insulin solution is prepared. Solutions of compoundsat different concentrations (8, 30 or 100 mg/mL) in water are prepared.An equivolume (50/50) mixture between the human insulin solution and thesolution of compound is prepared to lead to a solution containing 100IU/mL of human insulin and the desired concentration of compound (4, 15or 50 mg/mL). The pH of the various solutions is adjusted to pH 5.3 byadding 200 mM acetic acid.

The appearance of the solution is documented. If the solution is turbid,the compound at the test concentration does not allow dissolution of thehuman insulin. If the solution is translucent, the compound allowsdissolution of the human insulin at the test concentration. In this way,the concentration of compound required to dissolve the human insulin atits isoelectric point may be determined. The lower this concentration,the greater the affinity of the compound for human insulin.

The results obtained are presented in Table 3. The results show that thecompounds and the polysaccharides do not have the same properties interms of human insulin dissolution.

TABLE 3 Dissolution of Dissolution of Dissolution of human insulin humaninsulin human insulin Compounds at 100 IU/mL at 100 IU/mL at 100 IU/mL(examples) with the com- with the com- with the com- or Polysaccharidespound at 4 pound at 15 pound at 50 (counterexamples) mg/mL mg/mL mg/mLCounterexamples Polysaccharide 1 Yes Yes Yes Polysaccharide 4 Yes YesYes Polysaccharide 3 Yes Yes Yes Polysaccharide 2 Yes Yes YesPolysaccharide 5 Yes Yes Yes Examples Compound 1 No No Yes Compound 2 NoNo Yes Compound 3 No No Yes Compound 4 No No Yes Compound 6 No No YesCompound 8 No No Yes Compound 9 No No Yes Compound 10 No No Yes

E2. Dissolution of Lispro Insulin at its Isoelectric Point

Lispro insulin has an isoelectric point at 5.3. At this pH, lisproinsulin precipitates. A test demonstrating the formation of a complex oflispro insulin with the various compounds is performed at theisoelectric point. If an interaction exists, it is possible to dissolvethe lispro insulin at its isoelectric point.

The commercial formulation of lispro insulin (Humalog®) is dialyzedagainst 1 mM PO₄ buffer (pH 7). After dialysis, the lispro insulinconcentration is about 90 IU/mL. The lyophilized compound is weighed outand dissolved in the lispro insulin solution to lead to formulationscontaining lispro insulin at 90 IU/mL and the compound at the desiredconcentrations (4, 15 or 50 mg/mL). The pH of the various solutions isadjusted to pH 5.3 by adding 200 mM acetic acid.

The appearance of the solution is documented. If the solution is turbid,the compound at the test concentration does not allow dissolution of thelispro insulin. If the solution is translucent, the compound allowsdissolution of the lispro insulin at the test concentration. In thisway, the concentration of compound required to dissolve the lisproinsulin at its isoelectric point may be determined. The lower thisconcentration, the greater the affinity of the compound for the lisproinsulin.

The results obtained are presented in Table 4. The results show that thecompounds and the polysaccharides do not have the same properties interms of lispro insulin dissolution.

TABLE 4 Dissolution of Dissolution of Dissolution of lispro insulinlispro insulin lispro insulin Compounds at 90 IU/mL at 90 IU/mL at 90IU/mL (examples) with the com- with the com- with the com- orPolysaccharides pound at 4 pound at 15 pound at 50 (counterexamples)mg/mL mg/mL mg/mL Counterexamples Polysaccharide 1 Yes Yes YesPolysaccharide 3 Yes Yes Yes Polysaccharide 2 Yes Yes Yes ExamplesCompound 1 No No Yes Compound 2 No No Yes Compound 3 No No Yes

F Interaction with Albumin

F1: In order to determine the interactions between the variouspolysaccharides or compounds and a model protein such as albumin, aCentricon test (membrane with a cut-off threshold of 50 kDa) wasperformed. A solution of polysaccharide or of compound at 7.3 mg/mL wasdiluted three-fold in a solution of BSA (bovine serum albumin) at 20mg/mL in PBS (concentration in the mixture: 2.43 mg/mL of polysaccharideor of compound, 13.3 mg/mL of albumin and about 100 mM of salts).

This mixture was centrifuged on a Centricon to make about half thevolume pass through the membrane. The albumin is quantitatively retainedon the Centricon membrane. The polysaccharides and compounds analyzedalone pass in large majority through the membrane (for thepolysaccharides having the largest molar masses, about 20% of thepolysaccharide is retained).

After centrifugation, the polysaccharide or compound is assayed by UV inthe filtrate. The percentage of polysaccharide or compound bound to thealbumin is calculated via the following equation:

(1-[polysaccharide or compound in the filtrate in the presence ofalbumin]/[polysaccharide or compound in the filtrate in the absence ofalbumin])*100

The results obtained are presented in Table 5. It is very clearlyobserved that the polysaccharides of molar mass 5-15 kDa are stronglyretained by the albumin in this test. In contrast, the compounds of theinvention of lower molar mass are markedly less retained by the albuminin this test.

TABLE 5 % Polysaccharide or % Polysaccharide or Compound Compound boundto BSA Counterexamples Polysaccharide 4 97% Polysaccharide 1 95%Polysaccharide 3 77% Polysaccharide 5 86% Polysaccharide 2 82% ExamplesCompound 2 21% Compound 1 20% Compound 3 27% Compound 4 24% Compound 524% Compound 6 26% Compound 7 27% Compound 8 27% Compound 9 43% Compound11 35%

The invention claimed is:
 1. A rapid acting insulin lispropharmaceutical composition suitable for injection and having a pH of7.4±0.4, comprising: an insulin lispro in hexameric form; wherein theinsulin has a concentration between 100 to 200 IU/mL; a polyanioniccompound in a concentration between 10 and 30 mM, wherein thepolyanionic compound is citric acid or the Na⁺, K⁺, Ca²⁺ or Mg²⁺ saltthereof; a preservative; a tonicity agent; the insulin pharmaceuticalcomposition having a circular dichroism signal at 251 nm at or below−300 deg cm²dmol⁻¹.
 2. The rapid acting insulin lispro pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition does notinclude EDTA.
 3. The rapid acting insulin lispro pharmaceuticalcomposition of claim 1, wherein the tonicity agent is selected from thegroup consisting of glycerol, sodium chloride (NaCl), mannitol andglycine.
 4. The rapid acting insulin lispro pharmaceutical compositionof claim 3, wherein the tonicity agent is sodium chloride.
 5. The rapidacting insulin lispro pharmaceutical composition of claim 4, furthercomprising zinc salts.
 6. The rapid acting insulin lispro pharmaceuticalcomposition of claim 1, wherein the preservative is m-cresol.
 7. Therapid acting insulin lispro pharmaceutical composition of claim 5,further comprising a surfactant.